Protein encoded by a nucleic acid

ABSTRACT

A method is provided for identifying a compound that modulates a cellualr response associated with Homer and mediated by a cell-surface or an intracellular receptor. A method is further provided for identifying a compound that modulates receptor activated calcium mobilization associated with Homer. A method is provided for identifying a compound that inhibits Homer protein activity based on the crystal structure coordinates of Homer protein binding domain. A method is also provided for identifying a compound that affects the formation of cell surface receptors into clusters. Also provided are nucleic acids encoding Homer proteins as well as Homer proteins, and Homer interacting proteins.

RELATED APPLICATIONS

This application is a divisional application of U.S. application Ser.No. 10/192,381 filed Jul. 9, 2002, now issued as U.S. Pat. No.6,864,083; which is a divisional application of U.S. application Ser.No. 09/377,285 filed Aug. 18, 1999, now issued as U.S. Pat. No.6,720,175; which claims the benefit under 35 USC § 119(e) to U.S.application Ser. No. 60/138,494 filed Jun. 10, 1999, now abandoned, U.S.application Ser. No. 60/138,493 filed Jun. 10, 1999, now abandoned, U.S.application Ser. No. 60/138,426 filed Jun. 10, 1999, now abandoned andto U.S. application Ser. No. 60/097,334 filed Aug. 18, 1998, nowabandoned. The disclosure of each of the prior applications isconsidered part of and is incorporated by reference in the disclosure ofthis application.

STATEMENT AS TO FEDERALLY SPONSORED RESEARCH

This invention was made with Government support under Grant No. RO1DA10309, RO1 DA11742 and KO2 MH01152, awarded by the National Institutesof Health. The government may have certain rights in the invention.

FIELD OF THE INVENTION

The present invention relates generally to protein-protein interactionsand more specifically to molecules involved in mediatingreceptor-activated or ion channel-mediated intracellular calciummobilization or concentration.

BACKGROUND OF THE INVENTION

The mature central nervous system exhibits the capacity to altercellular interactions as a function of the activity of specific neuronalcircuits. This capacity is believed to underlie learning and memorystorage, age-related memory loss, tolerance to and dependence on drugsof abuse, recovery from brain injury, epilepsy as well as aspects ofpostnatal development of the brain (Schatz, C., Neuron, 5:745, 1990).Currently, the role of activity-dependent synaptic plasticity is bestunderstood in the context of learning and memory. Cellular mechanismsunderlying activity-dependent plasticity are known to be initiated byrapid, transmitter-induced changes in membrane conductance propertiesand activation of intracellular signaling pathways (Bliss andCollingridge, Nature, 361:31, 1993). Several lines of evidence alsoindicate a role for rapid synthesis of mRNA and protein in long-termneuroplasticity. For example, classical studies of learning and memorydemonstrate a requirement for protein synthesis in long-term, but notshort-term memory (Flexner, et al., Science, 141:57, 1963; Agranoff, B.,Basic Neurochemistry, 3rd Edition, 1981; Davis and Squire, Physiol.Bull., 96:518, 1984), and long-term enhancement of synapticconnectivity, studied in cultured invertebrate neurons (Montarolo, etal., Science, 234:1249, 1986; Bailey, et al., Neuron, 9:749, 1992) or inthe rodent hippocampus (Frey, et al., Science, 260:1661, 1993; Nguyen,et al., Science, 265:1104, 1994), is blocked by inhibitors of either RNAor protein synthesis. Importantly, inhibitors of macromolecularsynthesis are most effective when administered during a brief timewindow surrounding the conditioning stimulus indicating a specialrequirement for molecules that are rapidly induced (Goelet, et al.,Nature, 322:419, 1986).

Immediate early genes (IEGs) are rapidly induced in neurons byneurotransmitter stimulation and synaptic activity and are hypothesizedto be part of the macromolecular response required for long-termplasticity (Goelet, et al., supra; Sheng and Greenberg, Neuron, 4:477,1990; Silva and Giese, Neurobiology, 4:413, 1994). To identify cellularmechanisms that may contribute to long-term plasticity in the vertebratebrain, differential cloning techniques have been used to identify genesthat are rapidly induced by depolarizing stimuli (Nedivi, et al.,Nature, 363:713, 1993; Qian, et al., Nature, 361:453, 1993; Yamagata, etal., Neuron, 11:371, 1993; Yamagata, et al., Learning and Memory 1:140,1994; Yamagata, et al., Journal of Biological Chemistry, 269:16333,1994; Andreasson and Worley, Neuroscience, 69:781, 1995; Lyford, et al.,Neuron, 14:433, 1995). In contrast to the earlier focus on transcriptionfactors, many of the newly characterized IEGs represent molecules thatcan directly modify the function of cells and include growth factors(Nedivi, et al., supra; Andreasson and Worley, supra ), secreted enzymesthat can modify the extracellular matrix, such as tissue plasminogenactivator (Qian, et al., supra), enzymes involved in intracellularsignaling, such as prostaglandin synthase (Yamagata, et al., supra), anda novel homolog of H-Ras, termed Rheb (Yamagata, et al., supra), as wellas a novel cytoskeleton-associated protein, termed Arc (Lyford, et al.,supra). The remarkable functional diversity of this set of rapidresponse genes is representative of the repertoire of cellularmechanisms that are likely to contribute to activity-dependent neuronalplasticity.

Pharmaceutical agents often act by modulating signaling between cells orwithin cells. For example, Prozac alters the reuptake of theneurotransmitter serotonin and enhances aspects of its signalingfunction in brain. Nonsteroidal antiinflammatory drugs (NSAIDs) act byinhibiting the activity of cyclooxygenase enzyme, which is involved inthe signaling pathways of inflammation. Viagra modifies theintracellular guanylate cyclase response to autonomic neurotransmittersin erectile tissues. These, and other precedent setting pharmaceuticals,validate the notion that specific signaling pathways may be targeted fortherapeutic development.

Cellular mechanisms that modify important intracellular signals caninvolve changes in intracellular calcium. This type of mechanism is usedin brain neurons to adapt to changes in intercellular signaling, and isdemonstrated to exert powerful effects on cellular responses induced byglutamate. Similar, though distinct, cellular mechanism may be used tomodulate intracellular calcium signals in other tissues including heart,lung, liver and skeletal muscle. Compounds that can modify thismechanism can modulate natural transmitter signals and may exerttherapeutic effects.

Classical studies demonstrated that activation of receptors on the cellsurface evoke changes in the level of specific, diffusable moleculesinside the cell. The regulated production of these molecules serves tosignal events happening at the membrane surface to intracellularreceptors and are therefore termed second messenger signaling pathways.Major second messenger pathways include the phosphoinositide pathway,which regulates intracellular calcium; the adenylate cyclase pathway,which regulates levels of cyclic AMP; the guanylate cyclase pathway,which regulates levels of cGMP; and the nitric oxide pathway whichregulates NO.

The regulated release of intracellular calcium is essential to thefunction of all tissues. Each tissue possesses a distinct physiologythat is dependent on receptor/transmitter-regulated release ofintracellular calcium. For example, synaptic function is modulated inbrain neurons by glutamate receptor regulated release of intracellularcalcium. Contractility of cardiac and smooth muscle is also regulated byintracellular calcium. Recent reviews of the role of calcium signalingin cellular responses include: Berridge, Nature 386:759 (1997);Berridge, J. Physiol. (London) 499:291 (1997); Bootman et al., Cell91:367 (1997).

Recent studies demonstrate that molecules that function together insignaling networks are frequently clustered together in macromolecularcomplexes. For example, components of the MAP kinase pathway form acomplex of cytosolic kinases with their specific substrates (Davis, Mol.Reprod. Dev. 42:459 (1995)). Similarly, proteins such as AKAP functionas scaffolds for specific kinases and their substrates (Lester andScott, Recent Prog. Horm. Res. 52:409 (1997)). Recently, a multi-PDZcontaining protein was identified in Drosophila (termed InaD) thatcouples the membrane-associated, light-activated ion channel with itseffector enzymes (Tsunoda et al., Nature 388:243 (1997)). Thebiochemical consequence of this clustering is that the localconcentrations of molecules that convey the signals between proteins areas high as possible. Consequently, signaling takes place efficiently.The clustering activity of these proteins is essential to normalfunction of the signaling cascade (Lester and Scott, supra 1997; Tsunodaet al., supra 1997). Accordingly, Accordingly, agents that alter thesesignaling complexes will modify the response due to transmitter or otherform of cellular stimulation in a way that mimics more classicalreceptor agonists or antagonists. For example, a metabotropic glutamatereceptor signaling may be blocked either at the receptor by conventionalreceptor antagonists or by uncoupling the metabotropic receptor from itsintracellular IP3 receptor by agents that block the cross-linkingactivity of Homer family proteins.

The identification of molecules regulating the aggregation ofneurotransmitter receptors at synapses is central to understanding themechanisms of neural development, synaptic plasticity and learning. Themost well characterized model for the synaptic aggregation of ionotropicreceptors is the neuromuscular junction. Early work showed that contactbetween the axon of a motor neuron and the surface of a myotube rapidlytriggers the accumulation of preexisting surface acetylcholine receptors(Anderson and Cohen, J Physiol 268:757-773, 1977; Frank and Fischbach, JCell Biol 83:143-158, 1979). Subsequent work has shown that agrin, acomplex glycoprotein secreted by the presynaptic terminal, activates apostsynaptic signal transduction cascade (reviewed by Colledge andFroehner, Curr Opin Neurobiol 8:357-63, 1998), that leads to receptorclustering by the membrane associated protein rapsyn.

SUMMARY OF THE INVENTION

Homer proteins, the products of neuronal immediate early genes,selectively bind the carboxy-termini of certain cell-surface receptors(e.g., group 1 metabotropic receptors), certain intracellular receptorsand binding proteins (e.g., inositol trisphosphate receptors, ryanodinereceptor, Shank proteins, I42). Many forms of Homer proteins contain a“coiled-coil” structure in the carboxy-terminal domain which mediateshomo- and heteromultimerization between Homer proteins. The presentinvention is based on the seminal discovery that Homer plays asignificant role in mediating receptor-activated calcium mobilizationfrom internal stores and that Homer proteins regulate aspects ofreceptor clustering.

In one embodiment, a method is provided for identifying a compound thatmodulates a cellular response mediated by a cell-surface receptor. Themethod includes incubating a test compound and a cell expressing acell-surface receptor and a Homer protein under conditions sufficient topermit the compound to interact with the cell, and exposing the cell toa cell-surface receptor ligand. A cellular response to the ligand by thecell incubated with the compound is compared with a cellular response ofthe cell not incubated with the compound wherein a difference incellular response identify a compound that modulates a Homer-associatedcellular response.

In another embodiment, a method is provided for identifying a compoundthat modulates a cellular response mediated by an intracellularreceptor. The method includes incubating the compound, and a cellexpressing an intracellular receptor and a Homer protein underconditions sufficient to permit the compound to interact with the celland exposing the cell to conditions that activate the intracellularreceptor. A cellular response by a cell incubated with the compound iscompared with a cellular response of a cell not incubated with thecompound wherein a difference in a cellular response identifies acompound that modulates a Homer-associated cellular response.

In yet another embodiment, a method is provided for identifying acompound that modulates receptor activated calcium mobilization in acell. The method includes incubating the compound and a cell expressinga Homer protein under conditions sufficient to permit the compound tointeract with the cell and exposing the cell to conditions sufficient toactivate calcium mobilization. The receptor-activated calciummobilization of a cell incubated with said the compound is compared withthe receptor-activated calcium mobilization of a cell not incubated withthe compound wherein a difference in calcium mobilization is indicativeof an effect of the compound on Homer-associated calcium mobilization.

In another embodiment, a method is provided for modulatingreceptor-mediated calcium mobilization. The method includes exposing acell expressing Homer protein to a compound in a sufficient amount tomodulate the calcium mobilization that typically occurs when a cell isexposed to an amount of ligand sufficient to activate an intercellualrsignaling pathway that includes Homer protein.

In another embodiment, a method is provided for identifying a compoundthat inhibits Homer protein activity. The method includes identifying aninhibitor of Homer binding or crosslinking activity and identifying aninhibitor of Homer protein activity that forms covalent or non-covalentbonds with amino acids in a Homer protein binding site, based upon thecrystal structure coordinates of Homer protein binding domain. andsynthesizing the inhibitor.

In one embodiment, a method is provided for identifying a compound thataffects the formation of cell surface receptors into clusters. Themethod includes incubating the compound and a cell expressing a Homerprotein and a Homer interacting protein, e.g., a Shank protein, underconditions sufficient to allow the compound to interact with the celland determining the effect of the compound on the formation ofcell-surface receptors into clusters. The formation of cell-surfacereceptors into clusters of a cell contacted with the compound iscompared to the formation of cell-surface receptors into clusters of acell not contacted with the compound, wherein a difference in theformation of clusters is indicative of a compound that affects formationof cell surface receptors into clusters.

In another embodiment, a method is provided for treating a disorderassociated with glutamate receptors, including metabotropic andNMDA-type glutamate receptors, in a subject. The method includesadministering to a subject in need, a therapeutically effective amountof a compound that modulates Homer protein activity.

In another embodiment, a method is provided for treating a disorderassociated with Homer protein activity including administering to asubject in need a therapeutically effective amount of a compound thatmodulates Homer protein activity. The compound may be identified by amethod of the invention described herein.

In another embodiment, there is provided an isolated nucleic acidencoding Homer protein 1b, having the nucleotide sequence as set forthin SEQ ID NO:3 as well as an isolated Homer protein having substantiallythe same amino acid sequence as set forth in SEQ ID NO:4.

In another embodiment, there is provided an isolated nucleic acidencoding Homer protein 1c, as well as an isolated Homer protein.

In another embodiment, there is provided an isolated nucleic acidencoding Homer protein 2a, having the nucleotide sequence as set forthin SEQ ID NO:7 as well as an isolated Homer protein having substantiallythe same amino acid sequence as set forth in SEQ ID NO:8.

In another embodiment, there is provided an isolated nucleic acidencoding Homer protein 2b, having the nucleotide sequence as set forthin SEQ ID NO:9 as well as an isolated Homer protein having substantiallythe same amino acid sequence as set forth in SEQ ID NO:10.

In another embodiment, there is provided an isolated nucleic acidencoding Homer protein 3, having the nucleotide sequence as set forth inSEQ ID NO:11 as well as an isolated Homer protein having substantiallythe same amino acid sequence as set forth in SEQ ID NO:12.

In another embodiment, there is provided an isolated peptide having theamino acid sequence set forth in SEQ ID NO:13 an isolated peptide havingthe amino acid sequence set forth in SEQ ID NO:14.

In yet another embodiment, there is provided an isolated nucleic acidencoding Homer Interacting Protein, having the nucleotide sequence asset forth in SEQ ID NO:15 or 17 with a deduced amino acid sequence asset forth in SEQ ID NO:16 or 18, respectively.

-   -   In another embodiment, there is provided an isolated Homer        Interacting Protein having substantially the same amino acid        sequence as set forth in SEQ ID NO:20.

In another embodiment, there is provided an isolated Homer InteractingProtein having substantially the same amino acid sequence as set forthin SEQ ID NO:22.

In yet a further embodiment, there is provided a substantially purifiedpolypeptide containing a proline rich region that is specificallycapable of specifically binding to polypeptides of the Homer family.

In still another embodiment, there is provided a transgenic non-humananimal having a transgene that expresses a Homer protein, e.g., Homer 1a(SEQ ID NO:2), chromosomally integrated into the germ cells of theanimal.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Schematic Representation of EVH1 Domain-containing Proteins.EVH1 domains are found at or near the N-termini of Homer, Ena, Mena,VASP, and WASP proteins. Homer 1b/2/3 encode a CC domain which mediatesmultimerization between various Homer proteins. In ENA, Mena, VASP,WASP, and N-WASP, the EVH1 domain is followed by a central proline richregion of variable length. The proteins are drawn to the scale shown,and the respective amino acid lengths are shown at the right.

FIG. 2. Structure-Based Alignment of EVH1, PH, and PTB Domain Sequences.A structure-based sequence alignment between EVH1 domains and theθ-spectrin PH domain and the IRS-1 PTB domain is shown. Species areindicated by Rn (rat), Hs (human), Mm (mouse), and Dm (Drosophila).Elements of the Homer EVH1 domain secondary structure are represented byarrows (θ-strands), cylinders (I-helices), and lines (coils). Conservedresidues (among EVH1 domains) are highlighted. The fractional solventaccessibility (FAS) of each residue in Homer 1a is indicated by ovals.Filled ovals=0≦FAS≦0.1 (buried); shaded ovals=0.1<FAS≦0.4 (partiallyaccessible); open ovals=FAS>0.4. Mutations in the EVH1 domain of theWASP gene are indicated in lower case letters below the WASP amino acidsequence. Mutations that are associated with the severe WAS phenotypeare shown in bold letters (Zhu et al, 1997). Sites mutated to more thanone residue are indicated by asterisks. Bold asterisks indicate residuesthat, when mutated, affect the interaction of WASP with WIP (Stewart etal., 1999). Residues of Homer, θ-spectrin, and IRS-1 that align wellfollowing structural superposition and were used to calculate rmsdifferences in CI positions between these domains are underlined in theIRS-1 sequence. Gaps are indicated by dashes while continued sequencesat amino- and carboxy-termini are indicated by periods. Residuenumbering for Homer 1a is shown above its amino acid sequence. Thenumber of the last included residue of each protein is shown at the endof each row. Sequences shown are Homer 1a Rn (SEQ ID NO:63), Homer Dm(SEQ ID NO:64), Ena Dm (SEQ ID NO:65), Mena Mm (SEQ ID NO:66), EVL Mm(SEQ ID NO:67), SIF Dm (SEQ ID NO:68), VASP Hs (SEQ ID NO:69), N-WASP Hs(SEQ ID NO:70), WASP Hs (SEQ ID NO:71), WASP mut (SEQ ID NO:72), θ-specMm (SEQ ID NO:6), IRS-1 Hs (SEQ ID NO:5).

FIG. 3. Ribbon Diagram of the Homer 1a EVH1 Domain. The amino andcarboxy termini are indicated, and elements of secondary structure arelabeled to correspond to homologous structures in PH and PTB domains. Anadditional short region of θ-strand between θ1 and θ2 has been labeledθi.

FIG. 4. Structural Comparison of EVH1, PH, and PTB Domains. Ribbondiagrams (A)-(C) and surface representations (D)-(F) of the Homer 1EVH1, θ-spectrin PH, and IRS-1 PTB domains, respectively, are shown. Allmolecules are shown in a similar orientation, which is rotated about 45°about the vertical axis from orientations shown in FIG. 3. Theθ-spectrin PH domain is shown with bound inositol trisphosphate (Hyvonenet al, 1995). The IRS-1 domain is shown complexed to aphosphotyrosine-containing peptide derived from the insulin receptor(ECk et al., 1996).

FIG. 5. Versatile Ligand Recognition by PH-Like Domain. Sterodiagram ofa backbone trace of Homer 1 EVH1 doamin showing the relative positionsof IP3 as bound by the θ-spectrin and PLC-Λ PH domains, as well as thepeptide ligands for the IRS-1 and Numb PTB domains is shown. Theorientations of the EVH1 domain is similar to that in FIG. 4. Ligandpositions were determined by superimposing the backbone traces of theEVH1, PH and PTB domains in the program) (Jones et al., 1991).

FIG. 6. Mapping of WAS-Causing and Homer Binding Mutations on the EVH1Surface. (A) and (B) Surface representations of the Homer1 EVH1 doaminwith sites homologous to positions of WASP mutations (in parentheses)colored according to solvent accessibility. Solvent exposed residues areshown in magenta, and buried or partially buried residues are shown inblue. Residue assignments are based on the sequence shown in FIG. 2.WASP EVH1 mutations are listed in Table 2. Surface representations ofHomer 1 EVH1 domain showing the location of residues targeted bysite-directed mutagenesis. Mutations that disrupt binding of Homer EVH1to ligands in an in vitro binding assay are shown in red, while thosethat have no effect on binding are shown in light blue (see Table 3).The orientation of the EVH1 domain in panels A and C is identical tothat in FIG. 4A and D. IN panesl B and D., the moleucle is rotated about180 degrees about the vertical axis.

FIGS. 7 through 45 are described in the following table.

Figures Homer Family Proteins and Homer Interacting Proteins FIGURE SEQNo. ID No. Sequence 1 Human Homer 1a (nucleic acid) 7 2 Human Homer 1a(amino acid) 8 3 Human Homer 1b (nucleic acid) 9 4 Human Homer 1b (aminoacid) 2 5 IRS-1 2 6 β-spectrin 10 7 Human Homer 2a (nucleic acid) 11 8Human Homer 2a (amino acid) 12 9 Human Homer 2b (nucleic acid) 13 10Human Homer 2b (amino acid) 14 11 Human Homer 3 (nucleic acid) 15 12Human Homer 3 (amino acid) 13 peptide binding-core region: PPXXFR 14peptide binding-extended region: ALTPPSPFRD 16 15 Homer interactingprotein: rat I30 (nucleic acid) 17 16 Homer interacting protein: rat I30(amino acid) 18a-b 17 Homer interacting protein: rat I42 (nucleic acid)19 18 Homer interacting protein: rat I42 (amino acid) 20 19 Homerinteracting protein: human I30 (nucleic acid) 21 20 Homer interactingprotein: human I30 (amino acid) 22a-c 21 Homer interacting protein:human I42 (nucleic acid) 23 22 Homer interactin protein: human I42(amino acid) 24 23 Mouse Homer 1a (nucleic acid) 25 24 Mouse Homer 1a(amino acid) 26 25 Mouse Homer 1b (nucleic acid) 27 26 Mouse Homer 1b(amino acid) 28 27 Mouse Homer 2a (nucleic acid) 29 28 Mouse Homer 2a(amino acid) 30 29 Mouse Homer 2b (nucleic acid) 31 30 Mouse Homer 2b(amino acid) 32 31 Mouse Homer 3 (nucleic acid) 33 32 Mouse Homer 3(amino acid) 34a-c 33 Rat Homer 1a (nucleic acid) 35 34 Rat Homer 1a(amino acid) 36 35 Rat Homer 1b (nucleic acid) 37 36 Rat Homer 1b (aminoacid) 38a-b 37 Rat Homer 1c (nucleic acid) 39 38 Rat Homer 1c (aminoacid) 40a-d 39 Rat Shank 3a (nucleic acid) 41 40 Rat Shank 3a (aminoacid) 42 41 Human Homer 3a (nucleic acid) 43 42 Human Homer 3a (aminoacid) 44 43 Rat INADL partial nucleic acid sequence 45 44 Rat INADLpartial amino acid sequence

DETAILED DESCRIPTION OF THE INVENTION

Homer represents a family of proteins that selectively binds thecarboxy-terminus of group 1 metabotropic receptors and is enriched atexcitatory synapses (Brakeman et al., 1977). In the adult brain, Homeris rapidly and transiently induced by physiological synaptic stimulithat evoke ion-term potentiation in the hippocampus (Brakeman et al.,1997; Kato et al., 1997), and is also induced in the striatum bydopaminetic drugs of addiction (Brakeman et al., 1997). The first Homergene identified, now termed Homer 1a (Brakeman et al., Nature386:2284-288 (1997); GenBank Accession No. U92079), is a member of afamily of closely related Homer proteins that are constitutivelyexpressed in brain (Kato et al., 1998; Sun et al., 1998; Xiao et al.,1998). There are now three mammalian genes identified and at least sixdistinct transcripts expressed in brain (Xiao et al., 1998). All Homerfamily members, including Homer 1a, contain an amino-terminal region ofabout 110 amino acids that binds metabotropic glutamate receptors 1a and5 (mGluR1a and mGluR5) (Xiao et al., 1998). The region of Homer thatinteracts with mGluR1a or 5 is termed “EVH1 domain”, based on homologyto similar domains in a family of proteins that include DrosophilaEnabled (Gertler et al., 1996), mammalian VASP (Haffner et al., 1995)and the Wescott-Aldrige protein (WASP) (Ponting and Phillips, 1997;Symons et al., 1996). The EVH1 domain of Homer is conserved at a levelof about 80% between Drosophila, rodent and human (Xiao et al., 1998)The Homer family EVH1 domain also can bind to intracellular receptorssuch as the inositol trisphosphate receptor and dyamin III. Binding ofHomer proteins in the EVH1 region is mediated by an amino acid sequencemotif that is rich in proline residues.

To explore the proline-rich motif and its role in Homer interactions, adeletion mutation strategy was used. A 50-amino acid deletion at thecarboxy-terminal end of mGluR5 destroyed binding to Homer. By contrast,a 41 amino acid deletion of mGluR5 retained full binding activity. Theintervening sequence is proline rich and shares sequence similarity withthe previously described SH3 ligand sequence (Yu, 1994). A series ofpoint mutants based on the known structure-function relationship for SH3ligands was prepared and binding assays confirmed generalcharacteristics of SH3 ligand binding, but also demonstrated that thatthe Homer binding site is distinct in the positioning of critical aminoacids (Tu et al., 1998). A consensus for binding was determined to bePPXXFR (SEQ ID NO:11), consistent with the observation that mutation ofeither of the proline residues or the phenylalanine, or a change intheir relative position, interrupted binding. The arginine in the lastposition was preferred over other tested amino acids, but is notessential. Mutations were identically effective in interrupting bindingto each of the Homer family members including Homer 1a, 1b/c, 2a/b, 3and an EVH1 fragment (110 amino acids) of Homer 1. Thus, it wasconcluded that the interaction with mGluR5 was mediated by the HomerEVH1 domain.

To further explore Homer binding, mutations of mGluR5 were tested usinga 250 amino acid carboxy-terminal fragment of the receptor, which had anidentical effect on binding when placed in the full length mGluR5protein (Tu et al., 1998). This exquisite sensitivity of Homer bindingto changes in single amino acids within the Homer-ligand site wasconfirmed in other Homer-interacting proteins including mGluR1a (Tu etal., 1998), Shank (Tu et al., 1999), and I42 (see below). To furtherconfirm that the interaction was mediated by a direct interaction at theHomer-ligand site (as opposed to a secondary allosteric effect on aremote binding site), synthetic 10-mer peptides with either the wildtype, or F-to-R mutation were prepared. The wild type peptide blockedbinding of mGluR1a or mGluR5 to each of the Homer family members (Tu etal., 1998). Approximately half of the binding was blocked at a peptideconcentration of 3.4 micromolar. By contrast, the F-to-R mutant peptidedid not alter binding at concentrations as high as 340 micromolar.

Most forms of Homer protein encode a carboxy-terminal domain with a“coiled-coil” structure. This coiled-coil domain mediates homo- andheteromultermization between Homer proteins (Kato et al., 1998; Xiao etal., 1998) and such multimers can be identified in normal brain tissue(Xiao et al., 1998). Homer proteins are enriched in brain tissuefractions from postsynaptic densities and are localized at theultrastructural level to postsynaptic densities. Homer 1a differs fromthe other members of the Homer family in that Homer 1a is notconstitutively expressed and it does not contain a carboxy terminalcoiled-coil domain. Experimental data showing that Homer proteinsinteract with cell-surface receptors and with intracellular receptors,and form multimeric complexes with other Homer proteins indicates animportant role for Homer proteins in intracellular signaling.

An exemplary polynucleotide encoding a Homer protein is set forth as SEQID NO: 1. The term “polynucleotide”, “nucleic acid”, “nucleic acidsequence”, or “nucleic acid molecule” refers to a polymeric form ofnucleotides at least 10 bases in length. By “isolated polynucleotide” ismeant a polynucleotide that is not immediately contiguous with both ofthe coding sequences with which it is immediately contiguous (one on the5′ end and one on the 3′ end) in the naturally occurring genome of theorganism from which it is derived. The term therefore includes, forexample, a recombinant DNA which is incorporated into a vector; into anautonomously replicating plasmid or virus; or into the genomic DNA of aprokaryote or eukaryote, or which exists as a separate molecule (e.g., acDNA) independent of other sequences. The nucleotides of the inventioncan be ribonucleotides, deoxyribonucleotides, or modified forms ofeither nucleotide. A polynucleotide encoding Homer includes “degeneratevariants”, sequences that are degenerate as a result of the geneticcode. There are 20 natural amino acids, most of which are specified bymore than one codon. Therefore, all degenerate nucleotide sequences areincluded in the invention as long as the amino acid sequence of apolypeptide encoded by the nucleotide sequence of SEQ ID NO: 1 isfunctionally unchanged.

A nucleic acid molecule encoding Homer includes sequences encodingfunctional Homer polypeptides as well as functional fragments thereof.As used herein, the term “functional polypeptide” refers to apolypeptide which possesses biological function or activity which isidentified through a defined functional assay (e.g., EXAMPLE 3), andwhich is associated with a particular biologic, morphologic, orphenotypic alteration in the cell. The term “functional fragments ofHomer polypeptide,” refers to fragments of a Homer polypeptide thatretain a Homer activity, e.g., the ability to interact with cell-surfaceor intracellular receptors or mediate intracellular calciummobilization, and the like. Additionally, functional Homer fragments mayact as competitive inhibitors of Homer binding, for example,biologically functional fragments, for example, can vary in size from apolypeptide fragment as small as an epitope capable of binding anantibody molecule to a large polypeptide capable of participating in thecharacteristic induction or programming of phenotypic changes within acell.

A functional Homer polypeptide includes a polypeptide as set forth inSEQ ID NO:2 and conservative variations thereof. The terms “conservativevariation” and “substantially similar” as used herein denotes thereplacement of an amino acid residue by another, biologically similarresidue. Examples of conservative variations include the substitution ofone hydrophobic residue such as isoleucine, valine, leucine ormethionine for another, or the substitution of one polar residue foranother, such as the substitution of arginine for lysine, glutamic acidfor aspartic acid, or glutamine for asparagine, and the like. The terms“conservative variation” and “substantially similar” also include theuse of a substituted amino acid in place of an unsubstituted parentamino acid provided that antibodies raised to the substitutedpolypeptide also immunoreact with the unsubstituted polypeptide.

Also included are other Homer nucleic acid and amino acid sequences,including Homer 1b (SEQ ID NOS:3 and 4); Homer 1c; Homer 2a SEQ ID NOS:7and 8); Homer 2b (SEQ ID NOS:9 and 10); and Homer 3 (SEQ ID NOS:11 and12).

Cell-surface receptors are important intermediaries in intercellularsignaling. A “cell-surface receptor” is a protein, usually having atleast one binding domain on the outer surface of a cell where specificmolecules may bind to, activate, or block the cell surface receptor.Cell surface receptors usually have at least one extracellular domain, amembrane spanning region (“transmembrane”) and an intracellular domain.Activation of a cell-surface receptor can lead to changes in the levelsof various molecules inside the cell. Several types of cell-surfacereceptors have been identified in a variety of cell types, includingligand-gated receptors, ligand-gated channels, voltage-activatedreceptors, voltage-activated channels, ion channels and the like.

One class of cell-surface receptor is excitatory amino acid receptors(EAA receptors) which are the major class of excitatory neurotransmitterreceptors in the central nervous system. “EAA receptors” are membranespanning proteins that mediate the stimulatory actions of glutamate andpossibly other endogenous acidic amino acids. EAA are crucial for fastexcitatory neurotransmission and they have been implicated in a varietyof diseases including Alzheimer's disease, stroke schizophrenia, headtrauma and epilepsy. EAA have also been implicated in the process ofaging In addition, EAA are integral to the processes of long-termpotentiation, one of the synaptic mechanisms underlying learning andmemory. There are three main subtypes of EAA receptors: (1) themetabotropic or trans ACPD receptors; (2) the ionotropic NMDA receptors;and (3) the non-NMDA receptors, which include the AMPA receptors andkainate receptors.

Ionotropic glutamate receptors are generally divided into two classes:the NMDA and non-NMDA receptors. Both classes of receptors are linked tointegral cation channels and share some amino acid sequence homology.GluR1-4 are termed AMPA (I-amino-3-hydroxy-5-methylisoxazole-4-propionicacid) receptors because AMPA preferentially activates receptors composedof these subunits, while GluR5-7 are termed kainate receptors as theseare preferentially sensitive to kainic acid. Thus, an “AMPA receptor” isa non-NMDA receptor that can be activated by AMPA. AMPA receptorsinclude the GluR1-4 family, which form homo-oligomeric andhetero-oligomeric complexes which display different current-voltagerelations and Ca²⁺ permeability. Polypeptides encoded by GluR1-4 nucleicacid sequences can form functional ligand-gated ion channels. An AMPAreceptor includes a receptor having a GluR1, GluR2, GluR3 or GluR4subunit. NMDA receptor subtypes include class NR2B and NR2D, forexample.

Metabotropic glutamate receptors are divided into three groups based onamino acid sequence homology, transduction mechanism and bindingselectivity: Group I, Group II and Group III. Each Group of receptorscontains one or more types of receptors. For example, Group I includesmetabotropic glutamate receptors 1 and 5 (mGluR1 and mGluR5), Group IIincludes metabotropic glutamate receptors 2 and 3 (mGluR2 and mGluR3)and Group III includes metabotropic glutamate receptors 4, 6, 7 and 8(mGluR4, mGluR6, mGluR7 and mGluR8). Each mGluR type may be found inseveral subtypes. For example, subtypes of mGluR1 include mGluR1a,mGluR1b and mGluR1c.

Group I metabotropic glutamate receptors represent a family of sevenmembrane spanning proteins that couple to G-proteins and activatephospholipase C (Nakanishi, 1994). Members of the family include mGluR1and mGluR5. Activation of these receptors results in the hydrolysis ofmemberane phosphatidylinositol bisphosphate to diacylglycerol, whichactivates protein kinase C. and inositol trisphosphate, which in turnactivates the inositol trisphosphate receptor to release intracellularcalcium. (Aramori and Nakanishi, 1992; Joly et al., 1995 Kawabata etal., 1998)

Activation of a glutamate receptor on the cell surface results in acellular response. A “cellular response” is an event or sequence ofevents that singly or together are a direct or indirect response by acell to activation of a cell surface receptor. A “cellular response” isalso the blockade or activation of selective and non-selective cationchannels and potentiation or inhibition of other cell-surface receptorresponses. In addition, a “cellular response” may be the activation ofan intracellular signaling pathway, including the activation of allsteps or any one step in an intracellular signaling pathway.

An “intracellular signaling pathway” is a sequence of events thattransduces information about an extracellular event into a signal tointracellular receptors or effector molecules such as enzymes. One typeof intracellular signaling pathway is a second messenger signalingpathway. It may begin with the activation of receptors on the cellsurface, which activation evokes changes in the level of specific,diffusible molecules inside the cell. The regulated production of thesemolecules serves to signal events to the intracellular receptors and istherefore termed a second messenger signaling pathway. Major secondmessenger pathways include the adenylate cyclase pathway, whichregulates levels of cyclic AMP, the phosphoinositide pathway, whichregulates intracellular calcium, guanylate cyclase, which regulateslevels of cGMP, and the nitric oxide pathway, which regulates nitricoxide.

A cellular response mediated by cell surface receptors can also includecalcium mobilization. A compound can modulate cellular responsesmediated by cell surface receptors by inhibiting or potentiating therelease of calcium from intracellular stores. A compound increasescalcium mobilization by increasing the release of calcium fromintracellular stores. A compound decreases calcium mobilization byinhibiting of the release of calcium from intracellular stores.

Cell-surface receptors are known to mediate cellular responses. Methodsfor demonstrating cellular responses are well known in the art (e.g.electrophysiological and biochemical methods). (See Examples section foradditional methodology). A method is provided for identifying a compoundthat modulates a cellular response mediated by a cell-surface receptor.The method includes incubating the compound and a cell expressing acell-surface receptor and a Homer protein under conditions sufficient topermit the compound to interact with the cell. The cell may be any cellof interest, including but not limited to neuronal cells, glial cells,cardiac cells, bronchial cells, uterine cells, testicular cells, livercells, renal cells, intestinal cells, cells from the thymus and spleen,placental cells, endothelial cells, endocrine cells including thyroid,parathyroid, pituitary and the like, smooth muscle cells and skeletalmuscle cells. The cell is exposed to a cell-surface receptor ligand. A“cell surface receptor ligand” is a compound that binds to the bindingsite of the cell-surface receptor thereby initiating a sequence ofevents that singly or together embrace a “cellular response”. The effectof the compound on the cellular response is determined, either directlyor indirectly, and a cellular response is then compared with a cellularresponse of a control cell. A suitable control includes, but is notlimited to, a cellular response of a cell not contacted with thecompound. The term “incubating” includes conditions which allow contactbetween the test compound and the cell of interest. “Contacting” mayinclude in solution or in solid phase.

Compounds which modulate a cellular response can include peptides,peptidomimetics, polypeptides, pharmaceuticals, chemical compounds andbiological agents, for example. Antibodies, neurotropic agents,anti-epileptic compounds and combinatorial compound libraries can alsobe tested using the method of the invention. One class of organicmolecules, preferably small organic compounds having a molecular weightof more than 50 and less than about 2,500 Daltons. Candidate agentscomprise functional groups necessary for structural interaction withproteins, particularly hydrogen bonding, and typically include at leastan amine, carbonyl, hydroxyl or carboxyl group, preferably at least twoof the functional chemical groups. The candidate agents often comprisecyclical carbon or heterocyclic structures and/or aromatic orpolyaromatic structures substituted with one or more of the abovefunctional groups.

The test agent may also be a combinatorial library for screening aplurality of compounds. Compounds such as peptides identified in themethod of the invention can be further cloned, sequenced, and the like,either in solution of after binding to a solid support, by any methodusually applied to the isolation of a specific DNA sequence Moleculartechniques for DNA analysis (Landegren et al., Science 242:229-237,1988) and cloning have been reviewed (Sambrook et al., MolecularCloning: a Laboratory Manual, 2nd Ed.; Cold Spring Harbor LaboratoryPress, Plainview, N.Y., 1998, herein incorporated by reference).

Candidate compounds are obtained from a wide variety of sourcesincluding libraries of synthetic or natural compounds. For example,numerous means are available for random and directed synthesis of a widevariety of organic compounds and biomolecules, including expression ofrandomized oligonucleotides and oligopeptides. Alternatively, librariesof natural compounds in the form of bacterial, fungal, plant and animalextracts are available or readily produced. Additionally, natural orsynthetically produced libraries and compounds are readily modifiedthrough conventional chemical, physical and biochemical means, and maybe used to produce combinatorial libraries. Known pharmacological agentsmay be subjected to directed or random chemical modifications, such asacylation, alkylation, esterification, amidification, etc., to producestructural analogs. Candidate agents are also found among biomoleculesincluding, but not limited to: peptides, saccharides, fatty acids,steroids, purines, pyrimidines, derivatives, structural analogs orcombinations thereof.

A variety of other agents may be included in the screening assay. Theseinclude agents like salts, neutral proteins, e.g., albumin, detergents,etc. that are used to facilitate optimal protein-protein binding and/orreduce nonspecific or background interactions. Reagents that improve theefficiency of the assay, such as protease inhibitors, nucleaseinhibitors, antimicrobial agents and the like may be used. The mixtureof components are added in any order that provides for the requisitebinding. Incubations are performed at any suitable temperature,typically between 4 and 40° C. Incubation periods are selected foroptimum activity, but may also be optimized to facilitate rapidhigh-throughput screening. Typically between 0.1 and 10 h will besufficient.

In another embodiment, a method is provided for identifying a compoundthat modulates a cellular response mediated by an intracellularreceptor. An “intracellular receptor” is a protein that binds particularintracellular molecules. Intracelluar receptors include ryanodinereceptors and inositol trisphosphate receptors, for example, an“inositol trisphosphate receptor” is a receptor that binds the compoundinositol 1,4,5 trisphosphate, which is an important intracellular secondmessenger. Inositol 1,4,5 trisphosphate is released from phosphatidylinositol bisphosphate by the action of a specific phospholipase C enzyme(PLC) and binds to and activates a calcium channel in the endoplasmicreticulum (ER).

A compound can modulate a cellular response mediated by an intracellularreceptor by inhibiting or potentiating the release of calcium fromintracellular stores, for example, a compound increases calciummobilization by increasing the release of calcium from intracellularstores. A compound decreases calcium mobilization by inhibiting of therelease of calcium from intracellular stores.

The method of the invention includes incubating the compound and a cellexpressing an intracellular receptor and a Homer protein underconditions sufficient to permit the compound to interact with the cell,exposing the cell to conditions that activate said intracellularreceptor, and comparing a cellular response in a cell incubated withsaid compound with the response of a cell not incubated with saidcompound. Methods for determining cellular responses mediated byintracellular signals are well known to one of skill in the art (e.g.,biochemical assays) and provided in the Examples as well.

A method is also provided for identifying a compound that modulatesreceptor-activated calcium mobilization. The term “calcium mobilization”means a change in the amount or concentration of free calcium (Ca⁺²)sequestered in the endoplasmic reticulum, sarcoplasmic reticulum ormitochondria of a cell. The method includes incubating the compound anda cell expressing a Homer protein under conditions sufficient to permitthe compound to interact with the cell and exposing the cell toconditions sufficient to activate calcium mobilization. Then, thecellular response of the cell exposed to the compound is compared to thecellular response of a cell not exposed to the compound. A difference ina cellular response is indicative of a compound that modulatesreceptor-activated calcium mobilization in a cell.

In another embodiment of the invention, a method is provided formodulating receptor-mediated calcium mobilization in a cell includingexposing a cell to a compound in a sufficient amount to modulate thecalcium mobilization that normally occurs when a cell is exposed to anamount of ligand sufficient to activate an intracellular signalingpathway. Those of skill in the art will understand that “the calciummobilization that normally occurs” depends on the cell type and on theligand activating the intracellular pathway (Berridge, 1997 supra;Berridge, 1998 supra; Bootman, 1997 supra). Methods of measuring freecalcium flux are well known in the art (e.g., imaging methodology usingcalcium-sensitive dyes such as fura-2 and the like).

A ligand which activates the intracellular signaling pathway may be anagonist or antagonist of metabotropic glutamate receptors. The terms“agonist” and “antagonist” are meant to include compounds that bind tothe receptor and, respectively, activate or block activation of thereceptor. Known agonists of metabotropic glutamate receptors includeglutamate, quisqualate, Ibotenate, homocysteine sulfinate and theneurotoxin θ-N-methylamino-L-alanine. Antagonists of metabotropicglutamate receptors include MCPG. Known agonists of the NMDA typeglutamate receptor include glutamate and NMDA and known antagonistsinclude MK-801 and APV.

Another embodiment of the invention includes a method of identifying acompound that inhibits Homer protein activity. The method relies onfunctional properties of the Homer EVH1 and coiled-coil binding domainsthat can be used to establish high-throughput screens for molecules thatinfluence these and other functional properties of Homer family members.Homer protein activity may be blocked, partially or completely, byinterfering with a protein or other molecule in the intracellularsignaling pathway though which Homer proteins act. For example, Homeractivity can be modulated, for example, by modulating Homer proteinexpression, by modifying the activity of the Homer EVH1 domain, bymodification of the activity of the Homer CC domain, by modification ofHomer crosslinking activity, and the like. Homer activity can also bemodulated with by interfering with the expression or activity of HomerInteracting Protein I42, Homer Interacting Protein I30, NR2D, ACK-2,Shank proteins, ryanodine, inositol trisphosphate, and hInaD, and thelike.

Homer proteins function as a regulated adapter network that cross-linksinteracting proteins. Cross-linking is determined by the bindingproperties of the Homer EVH1 domain, which recognize a uniqueproline-rich ligand with a core sequence consensus of PPXXFR (SEQ IDNO:13). This Homer ligand is present in all identified proteins thatnaturally associate with Homer, and the ability of Homer proteins tobind can be disrupted by single amino acid changes in this motif.Cross-linking activity of Homer proteins has demonstrated effects onglutamate receptor signaling and this action is due to the formation ofsignaling complexes that link cell-surface receptors with intracellularreceptors. Cross-linking by Homer proteins may also have consequences onreceptor trafficking or other cellular functions of the interactingproteins.

Development of agents that modulate activity of the Homer EVH1 domain isfurthered by knowledge of the crystal structure of Homer protein. Themethod includes designing inhibitors of Homer protein that formnon-covalent bonds with amino acids in the Homer binding sites basedupon the crystal structure co-ordinates of Homer protein binding domain;synthesizing the inhibitor; and determining whether the inhibitorinhibits the activity of Homer protein.

The “Homer protein binding domain” is a conserved sequence of aminoacids in the amino-terminal region of the that interacts with otherproteins. All Homer proteins possess a conserved region of about 175amino acids at their amino-termini. The 110 terminal amino acids in thisregion interact with the carboxy-termini of other proteins, for examplemetabotropic glutamate receptors, inositol trisphosphate receptors,Shank, and the like. The carboxy-termini region of the proteins to whichthe Homer protein binding domain may bind usually contains an amino acidsequence that contains a high number of proline residues.

One aspect of the invention resides in the obtaining of crystals ofHomer protein of sufficient quality to determine the three dimensional(tertiary) structure of the protein by X-ray diffraction methods. Theknowledge obtained concerning Homer proteins may be used in thedetermination of the three dimensional structure of the binding domainof Homer proteins. The binding domain can also be predicted by variouscomputer models. Upon discovering the three-dimensional proteinstructure of the binding domain, small molecules which mimic thefunctional binding of Homer protein to its ligands can be designed andsynthesized This is the method of “rational” drug design. Anotherapproach to “rational” drug design is based on a lead compound that isdiscovered using high thoughput screens; the lead compound is furthermodified based on a crystal stucture of the binding regions of themolecule in question. Accordingly, another aspect of the invention is toprovide material which is a starting material in the rational design ofdrugs which mimic or prevents the action of Homer proteins.

The term “crystal structure coordinates” refers to mathematicalcoordinates derived from mathematical equations related to the patternsobtained on diffraction of a monochromatic beam of X-rays by the atoms(scattering centers) of a Homer protein molecule in crystal form. Thediffraction data are used to calculate an electron density map of therepeating unit of the crystal. The electron density maps are used toestablish the positions of the individual atoms within the unit cell ofthe crystal. The crystal structure coordinates of the Homer proteinbinding domain are obtained from a Homer protein crystal havingorthorhombic space group symmetry P2₁2₁2₁ with a=33.79, b=51.40, andc=66.30 Angstroms. The coordinates of the Homer protein binding domaincan also be obtained by means of computational analysis.

The term “selenomethione substitution refers to the method of producinga chemically modified form of the crystal of Homer. The Homer protein isexpressed by bacterial in meida that is depleted in methionine andsupplement in selenomethionine. Selenium is thereby incorporated intothe crystal in place of methionine sulfurs. The location(s) of seleniumare determined by X-ray diffraction analysis of the crystal. Thisinformation is used to generate the phase information used to constructthree-dimensional structure of the protein.

The term “heavy atom derivatization” refers to the method of producing achemically modified form of the crystal of Homer. A crystal is soaked ina solution containing heavy metal atom salts or organometalliccompounds, which can diffuse through the crystal and bind to the surfaceof the protein. The location(s) of the bound heavy metal atom(s) aredetermined by X-ray diffraction analysis of the soaked crystal. Thisinformation is used to generate the phase information used to constructthree-dimensional structure of the protein.

Those of skill in the art understand that a set of structure coordinatesdetermined by X-ray crystallography is not without standard error.

The term “unit cell” refers to the basic parallelipiped shaped block.The entire volume of a crystal may be constructed by regular assembly ofsuch blocks.

The term “space group” refers to the arrangement of symmetry elements ofa crystal.

The term “molecular replacement” refers to a method that involvesgenerating a preliminary model of an Homer crystal whose structurecoordinates are not known, by orienting and positioning a molecule whosestructure coordinates are known. Phases are then calculated from thismodel and combined with observed amplitudes to give an approximateFourier synthesis of the structure whose coordinates are known.

The crystal structure coordinates of Homer protein may be used to designcompounds that bind to the protein and alter its physical orphysiological properties in a variety of ways. The structure coordinatesof the protein may also be used to computationally screen small moleculedata bases for compounds that bind to the protein. The structurecoordinates of Homer mutants (e.g., missense mutations, deletionmutations, and the like, obtained by site-directed mutagenesis, byexposure to mutagenic agents, through selection of naturally occurringmutants, etc.) may also facilitate the identification of relatedproteins, thereby further leading to novel therapeutic modes fortreating or preventing Homer-mediated conditions. A potential inhibitoris designed to form hydrogen bonds with tryptophan²⁴, phenylalanine⁷⁴,threonine⁶⁶, threonine⁶⁸, glutamine⁷⁶, alanine⁷⁸, threonine⁷⁰, andvaline⁸⁵ of the Homer binding domain.

A method is also provided for identifying a compound that affects theformation of cell surface receptors into clusters. The method includesincubating the compound and a cell expressing a Homer protein and aHomer Interacting protein, such as a Shank protein, a Homer InteractingProtein, and the like, under conditions sufficient to allow the compoundto interact with the cell, determining the effect of the compound on theformation of cell-surface receptors into clusters, and comparing theformation of cell-surface receptors into clusters in cells contactedwith the compound with the formation of cell surface receptors intoclusters in cells not contacted with the compound.

Shank proteins are a novel family of proteins found at the postsynapticdensity (PSD) and which are capable of binding to other proteins. Shankproteins contain multiple protein interaction domains, including ankyrinrepeats, SH3 domain, PDZ domain, at least one proline rich domain and atleast one SAM domain. The PDZ domain of Shank mediates binding to thecarboxy-terminus of guanylate kinase associated protein (GKAP), and thisinteraction is important in neuronal cells for the synaptic localizationof Shank proteins. Shank proteins also interact with Homer proteins andtherefore Shank and Homer may serve as a protein bridge that linksspecific proteins that bind to Homer and specific proteins that bind toShank. Exemplary Shank proteins include Shank 1a, Shank 1b and Shank 3,and cortactin binding protein, and the like.

A compound can affect the formation of cell-surface receptors intoclusters by either stimulating the formation of cell-surface receptorsinto clusters or by inhibiting the recruitment of cell-surface receptorsinto clusters. When the effect is “inhibition”, cell-surface clusteringis decreased as compared with the level in the absence of the testcompound. When the effect is “stimulation”, cell-surface clustering isincreased as compared to a control in the absence of the test compound.

A method is further provided for treating a subject with a disorderassociated with metabotropic receptors or ion channel receptorscomprising administering to the subject a therapeutically effectiveamount of a compound that modulates Homer protein activity. In yetanother embodiment, a method is provided for treating a subject with adisorder associated with Homer protein activity, comprisingadministering to the subject a therapeutically effective amount of acompound that modulates Homer protein activity.

Essentially, any disorder that is etiologically linked to a glutamatereceptor, an inositol trisphosphate receptor, a ryanodine receptor, aShank protein, I42 (or other Homer interacting proteins) or to a Homerprotein could be considered susceptible to treatment with an agent thatmodulates Homer protein activity. The disorder may be a neuronal celldisorder. Examples of neuronal cell disorders include but are notlimited to Alzheimer's disease, Parkinson's disease, stroke, epilepsy,neurodegenerative disease, Huntington's disease, and brain or spinalcord injury/damage, including ischemic injury. The disorder may also bea disorder of a cardiac disorder, a disorder of musculature, a renaldisorder, a uterine disorder or a disorder of bronchial tissue. Thedisorder may be epilepsy, glutamate toxicity, a disorder of memory, adisorder of learning or a disorder of brain development.

Detection of altered (decreased or increased) levels of “Homer proteinactivity” can be accomplished by hybridization of nucleic acids isolatedfrom a cell of interest with a Homer polynucleotide of the invention.Analysis, such as Northern Blot analysis, are utilized to quantitateexpression of Homer, such as to measure Homer transcripts. Otherstandard nucleic acid detection techniques will be known to those ofskill in the art. Detection of altered levels of Homer can alsoaccomplished using assays designed to detect Homer polypeptide. Forexample, antibodies or petides that specifically bind a Homerpolypeptide can be utilized. Analyses, such as radioimmune assay orimmunohistochemistry, are then used to measure Homer, such as to measureprotein concentration qualitatively or quantitatively.

Treatment can include modulation of Homer activity by administration ofa therapeutically effective amount of a compound that modulates Homer orHomer protein activity. The term “modulate” envisions the suppression ofHomer activity or expression when Homer is overexpressed or has anincreased activity as compared to a control. The term “modulate” alsoincludes the augmentation of the expression of Homer when it isunderexpressed or has a decreased activity as compared to a control. Theterm “compound” as used herein describes any molecule, e.g., protein,nucleic acid, or pharmaceutical, with the capability of altering theexpression of Homer polynucleotide or activity of Homer polypeptide.Treatment may inhibit the interaction of the EVH1 domain of Homer withits target protein, may increase the avidity of this interaction bymeans of allosteric effects, may block the binding activity of thecoiled-coil doamin of Homer or influence other functional properties ofHomer proteins.

Candidate agents include nucleic acids encoding a Homer, or thatinterfere with expression of Homer, such as an antisense nucleic acid,ribozymes, and the like. Candidate agents also encompass numerouschemical classes wherein the agent modulates Homer expression oractivity.

Where a disorder is associated with the increased expression of Homer,nucleic acid sequences that interfere with the expression of Homer canbe used. In this manner, the coupling of cell-surface and intracellularreceptors can be inhibited. This approach also utilizes, for example,antisense nucleic acid, ribozymes, or triplex agents to blocktranscription or translation of Homer mRNA, either by masking that mRNAwith an antisense nucleic acid or triplex agent, or by cleaving it witha ribozyme in disorders associated with increased Homer. Alternatively,a dominant negative form of Homer polypeptide could be administered.

When Homer is overexpressed, candidate agents include antisense nucleicacid sequences. Antisense nucleic acids are DNA or RNA molecules thatare complementary to at least a portion of a specific mRNA molecule(Weintraub, 1990, Scientific American, 262:40). In the cell, theantisense nucleic acids hybridize to the corresponding mRNA, forming adouble-stranded molecule. The antisense nucleic acids interfere with thetranslation of the mRNA, since the cell will not translate a mRNA thatis double-stranded. Antisense oligomers of about 15 nucleotides arepreferred, since they are easily synthesized and are less likely tocause problems than larger molecules when introduced into the targetcell. The use of antisense methods to inhibit the in vitro translationof genes is well known in the art (Marcus-Sakura, 1988, Anal.Biochem.,172:289).

Use of an oligonucleotide to stall transcription is known as the triplexstrategy since the oligomer winds around double-helical DNA, forming athree-strand helix. Therefore, these triplex compounds can be designedto recognize a unique site on a chosen gene (Maher, et al., 1991,Antisense Res. and Dev., 1(3):227; Helene, C., 1991, Anticancer DrugDesign, 6(6):569).

Ribozymes are RNA molecules possessing the ability to specificallycleave other single-stranded RNA in a manner analogous to DNArestriction endonucleases. Through the modification of nucleotidesequences which encode these RNAs, it is possible to engineer moleculesthat recognize specific nucleotide sequences in an RNA molecule andcleave it (Cech, 1988, J.Amer.Med. Assn., 260:3030). A major advantageof this approach is that, because they are sequence-specific, only mRNAswith particular sequences are inactivated.

There are two basic types of ribozymes namely, tetrahymena-type(Hasselhoff, 1988, Nature, 334:585) and “hammerhead”-type.Tetrahymena-type ribozymes recognize sequences which are four bases inlength, while “hammerhead”-type ribozymes recognize base sequences 11-18bases in length. The longer the recognition sequence, the greater thelikelihood that the sequence will occur exclusively in the target mRNAspecies. Consequently, hammerhead-type ribozymes are preferable totetrahymena-type ribozymes for inactivating a specific mRNA species and18-based recognition sequences are preferable to shorter recognitionsequences.

When a disorder is associated with the decreased expression of Homer,nucleic acid sequences that encode Homer can be used. An agent whichmodulates Homer expression includes a polynucleotide encoding apolypeptide of SEQ ID NO:2, 4, 8, 10 or 12, or a conservative variantthereof. Alternatively, an agent of use with the subject inventionincludes agents that increase the expression of a polynucleotideencoding Homer or an agent that increases the activity of Homerpolypeptide.

In another embodiment of the invention, there is provided a transgenicnon-human animal having a transgene that expresses Homer 1achromosomally integrated into the germ cells of the animal. Animals arereferred to as “transgenic” when such animal has had a heterologous DNAsequence, or one or more additional DNA sequences normally endogenous tothe animal (collectively referred to herein as “transgenes”)chromosomally integrated into the germ cells of the animal. Thetransgenic animal (including its progeny) will also have the transgenefortuitously integrated into the chromosomes of somatic cells.

Various methods to make the transgenic animals of the subject inventioncan be employed. Generally speaking, three such methods may be employed.In one such method, an embryo at the pronuclear stage (a “one cellembryo”) is harvested from a female and the transgene is microinjectedinto the embryo, in which case the transgene will be chromosomallyintegrated into both the germ cells and somatic cells of the resultingmature animal. In another such method, embryonic stem cells are isolatedand the transgene incorporated therein by electroporation, plasmidtransfection or microinjection, followed by reintroduction of the stemcells into the embryo where they colonize and contribute to the germline. Methods for microinjection of mammalian species is described inU.S. Pat. No. 4,873,191. In yet another such method, embryonic cells areinfected with a retrovirus containing the transgene whereby the germcells of the embryo have the transgene chromosomally integrated therein.When the animals to be made transgenic are avian, because avianfertilized ova generally go through cell division for the first twenty hin the oviduct, microinjection into the pronucleus of the fertilized eggis problematic due to the inaccessibility of the pronucleus. Therefore,of the methods to make transgenic animals described generally above,retrovirus infection is preferred for avian species, for example asdescribed in U.S. Pat. No. 5,162,215. If microinjection is to be usedwith avian species, however, a recently published procedure by Love etal., (Biotechnology, 12, Jan. 1994) can be utilized whereby the embryois obtained from a sacrificed hen approximately two and one-half h afterthe laying of the previous laid egg, the transgene is microinjected intothe cytoplasm of the germinal disc and the embryo is cultured in a hostshell until maturity. When the animals to be made transgenic are bovineor porcine, microinjection can be hampered by the opacity of the ovathereby making the nuclei difficult to identify by traditionaldifferential interference-contrast microscopy. To overcome this problem,the ova can first be centrifuged to segregate the pronuclei for bettervisualization.

The “non-human animals” of the invention are murine typically (e.g.,mouse). The “transgenic non-human animals” of the invention are producedby introducing “transgenes” into the germline of the non-human animal.Embryonal target cells at various developmental stages can be used tointroduce transgenes. Different methods are used depending on the stageof development of the embryonal target cell. The zygote is the besttarget for microinjection. The use of zygotes as a target for genetransfer has a major advantage in that in most cases the injected DNAwill be incorporated into the host gene before the first cleavage(Brinster et al., Proc. Natl. Acad. Sci. USA 82:4438-4442, 1985). As aconsequence, all cells of the transgenic non-human animal will carry theincorporated transgene. This will in general also be reflected in theefficient transmission of the transgene to offspring of the foundersince 50% of the germ cells will harbor the transgene.

The term “transgenic” is used to describe an animal which includesexogenous genetic material within all of its cells. A “transgenic”animal can be produced by cross-breeding two chimeric animals whichinclude exogenous genetic material within cells used in reproduction.Twenty-five percent of the resulting offspring will be transgenic i.e.,animals which include the exogenous genetic material within all of theircells in both alleles. 50% of the resulting animals will include theexogenous genetic material within one allele and 25% will include noexogenous genetic material.

In the microinjection method useful in the practice of the subjectinvention, the transgene is digested and purified free from any vectorDNA e.g. by gel electrophoresis. It is preferred that the transgeneinclude an operatively associated promoter which interacts with cellularproteins involved in transcription, ultimately resulting in constitutiveexpression. Promoters useful in this regard include those fromcytomegalovirus (CMV), Moloney leukemia virus (MLV), and herpes virus,as well as those from the genes encoding metallothionin, skeletal actin,P-enolpyruvate carboxylase (PEPCK), phosphoglycerate (PGK), DHFR, andthymidine kinase. Promoters for viral long terminal repeats (LTRs) suchas Rous Sarcoma Virus can also be employed. Constructs useful in plasmidtransfection of embryonic stem cells will employ additional regulatoryelements well known in the art such as enhancer elements to stimulatetranscription, splice acceptors, termination and polyadenylationsignals, and ribosome binding sites to permit translation.

Retroviral infection can also be used to introduce transgene into anon-human animal, as described above. The developing non-human embryocan be cultured in vitro to the blastocyst stage. During this time, theblastomeres can be targets for retro viral infection (Jaenich, R., Proc.Natl. Acad. Sci USA 73:1260-1264, 1976). Efficient infection of theblastomeres is obtained by enzymatic treatment to remove the zonapellucida (Hogan, et al. (1986) in Manipulating the Mouse Embryo, ColdSpring Harbor Laboratory Press, Cold Spring Harbor, N.Y.). The viralvector system used to introduce the transgene is typically areplication-defective retro virus carrying the transgene (Jahner, etal., Proc. Natl. Acad. Sci. USA 82:6927-6931, 1985; Van der Putten, etal., Proc. Natl. Acad. Sci USA 82:6148-6152, 1985). Transfection iseasily and efficiently obtained by culturing the blastomeres on amonolayer of virus-producing cells (Van der Putten, supra; Stewart, etal., EMBO J. 6:383-388, 1987). Alternatively, infection can be performedat a later stage. Virus or virus-producing cells can be injected intothe blastocoele (D. Jahner et al., Nature 2-98:623-628, 1982). Most ofthe founders will be mosaic for the transgene since incorporation occursonly in a subset of the cells which formed the transgenic nonhumananimal. Further, the founder may contain various retro viral insertionsof the transgene at different positions in the genome which generallywill segregate in the offspring. In addition, it is also possible tointroduce transgenes into the germ line, albeit with low efficiency, byintrauterine retroviral infection of the midgestation embryo (D. Jahneret al., supra).

A third type of target cell for transgene introduction is the embryonalstem cell (ES). ES cells are obtained from pre-implantation embryoscultured in vitro and fused with embryos (M. J. Evans et al. Nature292:154-156, 1981; M. O. Bradley et al., Nature 309: 255-258, 1984;Gossler, et al., Proc. Natl. Acad. Sci USA 83: 9065-9069, 1986; andRobertson et al., Nature 322:445-448, 1986). Transgenes can beefficiently introduced into the ES cells by DNA transfection or by retrovirus-mediated transduction. Such transformed ES cells can thereafter becombined with blastocysts from a nonhuman animal. The ES cellsthereafter colonize the embryo and contribute to the germ line of theresulting chimeric animal. (For review see Jaenisch, R., Science 240:1468-1474, 1988).

“Transformed” means a cell into which (or into an ancestor of which) hasbeen introduced, by means of recombinant nucleic acid techniques, aheterologous nucleic acid molecule. “Heterologous” refers to a nucleicacid sequence that either originates from another species or is modifiedfrom either its original form or the form primarily expressed in thecell.

“Transgene” means any piece of DNA which is inserted by artifice into acell, and becomes part of the genome of the organism (i.e., eitherstably integrated or as a stable extrachromosomal element) whichdevelops from that cell. Such a transgene may include a gene which ispartly or entirely heterologous (i.e., foreign) to the transgenicorganism, or may represent a gene homologous to an endogenous gene ofthe organism. Included within this definition is a transgene created bythe providing of an RNA sequence which is transcribed into DNA and thenincorporated into the genome. The transgenes of the invention includeDNA sequences which encode Homer protein-sense and antisensepolynucleotides, which may be expressed in a transgenic non-humananimal. The term “transgenic” as used herein additionally includes anyorganism whose genome has been altered by in vitro manipulation of theearly embryo or fertilized egg or by any transgenic technology to inducea specific gene knockout. As used herein, the term “transgenic” includesany transgenic technology familiar to those in the art which can producean organism carrying an introduced transgene or one in which anendogenous gene has been rendered non-functional or “knocked out”.

Antibodies of the invention may bind to Homer proteins or Homerinteracting proteins provided by the invention to prevent normalinteractions of the Homer proteins and Homer Interacting proteins.Binding of antibodies to Homer proteins or Homer Interacting Proteinscan interfere with cell-signaling by interfering with an intracellularsignaling pathway. Binding of antibodies can interfere with Homerprotein binding to extracellular receptors, e.g., to NMDA receptors, tometabotropic receptors, and the like. Binding of antibodies caninterfere with Homer protein binding to intracellular receptors, e.g.,inositol trisphosphate receptors, and the like. Furthermore, binding toHomer proteins or to Homer Interacting Proteins can interfere withcell-surface receptor clustering mediated by Homer family proteins.

The antibodies of the invention can be used in any subject in which itis desirable to administer in vitro or in vivo immunodiagnosis orimmunotherapy. The antibodies of the invention are suited for use, forexample, in immunoassays in which they can be utilized in liquid phaseor bound to a solid phase carrier. In addition, the antibodies in theseimmunoassays can be detectably labeled in various ways. Examples oftypes of immunoassays which can utilize antibodies of the invention arecompetitive and non-competitive immunoassays in either a direct orindirect format. Examples of such immunoassays are the radioimmunoassay(RIA) and the sandwich (immunometric) assay. Detection of the antigensusing the antibodies of the invention can be done utilizing immunoassayswhich are run in either the forward, reverse, or simultaneous modes,including immunohistochemical assays on physiological samples. Those ofskill in the art will know, or can readily discern, other immunoassayformats without undue experimentation.

The term “antibody” as used in this invention includes intact moleculesas well as fragments thereof, such as Fab, F(ab′)2, and Fv which arecapable of binding to an epitopic determinant present in an inventionpolypeptide. Such antibody fragments retain some ability to selectivelybind with its antigen or receptor.

Methods of making these fragments are known in the art. (See forexample, Harlow and Lane, Antibodies: A Laboratory Manual, Cold SpringHarbor Laboratory, New York (1988), incorporated herein byreference).Monoclonal antibodies are made from antigen containingfragments of the protein by methods well known to those skilled in theart (Kohler, et al., Nature, 256:495, 1975).

Antibodies which bind to an invention polypeptide of the invention canbe prepared using an intact polypeptide or fragments containing smallpeptides of interest as the immunizing antigen. For example, it may bedesirable to produce antibodies that specifically bind to the N- orC-terminal domains of an invention polypeptide. The polypeptide orpeptide used to immunize an animal is derived from translated cDNA orchemically synthesized and can be conjugated to a carrier protein, ifdesired. Commonly used carrier proteins which may be chemically coupledto the immunizing peptide include keyhole limpet hemocyanin (KLH),thyroglobulin, bovine serum albumin (BSA), tetanus toxoid, and the like.

Invention polyclonal or monoclonal antibodies can be further purified,for example, by binding to and elution from a matrix to which thepolypeptide or a peptide to which the antibodies were raised is bound.Those of skill in the art will know of various techniques common in theimmunology arts for purification and/or concentration of polyclonalantibodies, as well as monoclonal antibodies (See, for example, Coligan,et al., Unit 9, Current Protocols in Immunology, Wiley Interscience,1994, incorporated by reference).

The antibodies of the invention can be bound to many different carriersand used to detect the presence of an antigen comprising thepolypeptides of the invention. Examples of well-known carriers includeglass, polystyrene, polypropylene, polyethylene, dextran, nylon,amylases, natural and modified celluloses, polyacrylamides, agaroses andmagnetite. The nature of the carrier can be either soluble or insolublefor purposes of the invention. Those skilled in the art will know ofother suitable carriers for binding antibodies, or will be able toascertain such, using routine experimentation.

There are many different labels and methods of labeling known to thoseof ordinary skill in the art. Examples of the types of labels which canbe used in the present invention include enzymes, radioisotopes,fluorescent compounds, colloidal metals, chemiluminescent compounds,phosphorescent compounds, and bioluminescent compounds. Those ofordinary skill in the art will know of other suitable labels for bindingto the antibody, or will be able to ascertain such, using routineexperimentation.

Another technique which may also result in greater sensitivity consistsof coupling the antibodies to low molecular weight haptens. Thesehaptens can then be specifically detected by means of a second reaction.For example, it is common to use such haptens as biotin, which reactswith avidin, or dinitrophenyi, puridoxal, and fluorescein, which canreact with specific antihapten antibodies.

In using the monoclonal and polyclonal antibodies of the invention forthe in vivo detection of antigen, e.g., Homer, the detectably labeledantibody is given a dose which is diagnostically effective. The term“diagnostically effective” means that the amount of detectably labeledantibody is administered in sufficient quantity to enable detection ofthe site having the antigen comprising a polypeptide of the inventionfor which the antibodies are specific.

The concentration of detectably labeled antibody which is administeredshould be sufficient such that the binding to those cells having thepolypeptide is detectable compared to the background. Further, it isdesirable that the detectably labeled antibody be rapidly cleared fromthe circulatory system in order to give the best target-to-backgroundsignal ratio.

As a rule, the dosage of detectably labeled antibody for in vivotreatment or diagnosis will vary depending on such factors as age, sex,and extent of disease of the individual. Such dosages may vary, forexample, depending on whether multiple injections are given, antigenicburden, and other factors known to those of skill in the art.

The following examples are intended to illustrate but not to limit theinvention in any manner, shape, or form, either explicitly orimplicitly. While they are typical of those that might be used, otherprocedures, methodologies, or techniques known to those skilled in theart may alternatively be used.

EXAMPLES

Homer 1a is an IEG and is the original member of a family of proteinsthat function together as a regulated adapter system that ishypothesized to control the coupling of membrane receptors tointracellular pools of releasable calcium. Homer proteins function atexcitatory synapses to couple membrane group 1 metabotropic glutamatereceptors (mGluR) to endoplasmic reticulum-associated inositoltrisphosphate receptors (IP3R) (Brakeman et al., 1997; Tu et al., 1998;Xiao eta!., 1998). Current studies suggest a broader role for Homerproteins in calcium signaling and receptor trafficking. The Shank familyof proteins was identified based on their association with Homer(Naisbitt et al., 1999; Tu et al., 1999). Shank, together with Homer,appears to be part of both the NMDA and group 1 mGluR signalingcomplexes. By virtue of its interaction with Shank, Homer provides amechanism to couple NMDA Ca2+ influx to intracellular Ca2+-induced Ca2+release pools. The inventors have identified additionalHomer-interacting proteins that provide insight into the role of Homerin trafficking of group 1 mGluR (e.g., SEQ ID NOS: 16, 18, 20, 22).Because these Homer-dependent cellular processes are regulated by theIEG form of Homer (Homer 1a), mechanisms by which Homer proteins canmodulate Ca2+ dynamics of mGluR and NMDA receptors, as well as regulatereceptor trafficking are defined.

Homer family proteins possess an N-terminal EVH1 domain that mediatesinteractions with mGluRs, IP3R, Shank and other novel proteins. The EVH1domain has been determined to bind the proline rich motif PPXXFR (SEQ IDNO:13) (Tu et al., 1998). The present invention provides the crystalstructure of the Homer EVH1 domain. In complementary studies, geneticapproaches were used to identify critical residues in both the EVH1domain and the ligand that modulate the affinity of the Homer-mGluR (andother Homer-interacting proteins) interaction. This information isessential to an understanding of the integrative cellular actions ofHomer proteins,. Together, these studies define the molecular basis ofspecificity of EVH1 interaction with its ligands, and provide insightinto how the EVH1 interaction is regulated.

This patent application includes a description of severalHomer-interacting proteins that are part of the signaling network thatis controlled by Homer (e.g., SEQ ID NOS: 16, 18, 20 and 22). Yeasttwo-hybrid screens and searches of NCBI protein data bases identified aset of known and novel candidate interacting proteins for Homer includethe ryanodine receptor, NMDA receptor subunit NR2D, human InaD and novelinteracting proteins termed I42 and I30. As described below, currentdata indicate that agents can be developed that specifically modulatethe crosslinking activity of Homer for these various receptors andthereby provide novel therapeutics that regulate the output of thesereceptors on cellular function.

Homer acts in several ways to regulate cellular function. Homer andHomer-related proteins function as an adapter system to couple membranereceptors to intracellular pools of releasable Ca. This “signaling”function of Homer is documented in Xiao et (1998), Tu et al (1998 and1999) Naisbett et al. (1999), as well as by studies of the novelHomer-interacting protein termed I42 (see below). By virtue of itscrosslinking activity, Homer proteins play a role in synaptogenesis andspatial targeting/trafficking of GluRs to other postsynaptic structuralproteins. This function of Homer is supported by observations in Tu etal (1999) and Naisbett et al (1999).

Initial Cloning of Homer; a Novel Brain Immediate Early Gene (IEG)

Homer was cloned in a differential screen of seizure-stimulatedhippocampus. Prior work, in which IEG induction was examined in brainprovided a detailed understanding of time course and tissue distributionof the IEG response (Cole et al., 1989; Saffen et al., 1988; Worley etal., 1990), and suggested a paradigm to maximally induce novel IEG mRNAs(Lanahan and Worley, 1998; Worley et al., 1990). Once cloned, in situhybridization was used to screen for IEGs that were regulated in otherparadigms that activate neurons including LTP stimulation in thehippocampus (Brakeman et al., 1997) and acute administration of cocaine.In these models, Homer was one of the most highly induced of all of theIEGs (Brakeman et al., 1997). Initial characterization of Homer waschallenging in that the mRNA was nearly 7 kb, while the best deducedopen reading frame was only 186 aa, and was located near the 5′ end ofthe cDNA (Brakeman et al., 1997). The 3′ UTR was over 5 kb. The ORF wasconfirmed by in vitro transcription and translation of the cDNA, andrabbit polyclonal antisera were generated against bacterially expressedfusion proteins. With these antibodies, we were able to demonstrate thatthe protein was rapidly and transiently induced in the hippocampusfollowing a seizure (Brakeman et al., 1997). This confirmed the deducedORF and assured us that the cDNA was indeed translated in brain.

Homer Selectively Binds Group 1 Metabotropic Receptors and is Enrichedat Synapses

In an effort to discover the function of Homer, a yeast 2-hybridtechnique (Chevray and Nathans, 1992; Fields and Song, 1989) was used toscreen a cDNA library prepared from rat hippocampus and cortex. The fulllength Homer IEG was used as bait. Among ˜30 confirmed interactingcDNAs, one encoded the C-terminal 250 aa of mGluR5. We initiallyconfirmed that the proteins bind using a GSTHomer in a pulldown assaywith either fragments of mGluR5, or full length mGluR5 expressed inheterologous cells (HEK293 cells) (Brakeman et al., 1997). Homer proteinalso bound to mGluR1a, but not mGluR2, 3, 4, or 7. This was aninteresting clue to the function of Homer since mGluR1 and mGluR5(termed group 1 metabotropic receptors) couple to phospholipase C andactive hydrolysis of phosphoinositides to generate inositoltrisphosphate and diacylglycerol (Nakanishi et al., 1994). mGluR1a and 5also share sequence similarity in their long, cytosolically disposedC-terminus. Other metabotropic glutamate receptors (termed group 2 and3) inhibit adenylate cyclase activity, and have short C-termini thatlack homology to group 1 receptors. We proceeded to test whether Homerand mGluR5 naturally associate in brain and confirmed that theseproteins co-immunoprecipitate from detergent extracts of hippocampus(Brakeman et al., 1997). The next major clue was provided by theobservation that Homer immunoreactivity was enriched at excitatorysynapses (Brakeman et al., 1997). In brain, Homer protein was associatedwith dendrites and showed a punctate pattern consistent with alocalization in spines. The binding properties and cellular distributionof Homer suggested a role at the excitatory synapse.

Homer is a Member of a Family of Closely Related Proteins that areEnriched at the Excitatory Synapse

A search of the NCBI sequence data base identified several ESTs thatshowed strong homology to Homer, but were clearly distinct in that theyencoded additional C-terminal sequence (Brakeman et al., 1997). Using acombination of screening strategies, a family of 12 cDNAs was identifiedfrom rat, mouse, Drosophila, and human (Xiao et al., 1998). All of thesecDNAs encoded proteins with a similar protein structure and were deducedto be the products of 3 independent mammalian genes (termed Homer 1, 2,3) and 1 Drosophilia gene. Like Homer IEG (now termed Homer 1a), all newfamily members contain an N-terminal, ˜110 amino acid domain that bindsmGluR1a/5 ((Xiao et al., 1998). The region of Homer that interacts withmGluR1a/5 is termed an EVH1 domain based on its modest homology (20-25%identity) to domains in a family of proteins that include DrosophiliaEnabled Gertler, 1996, mammalian VASP (Haffner et al., 1995) and theWiscott-Aldridge protein (WASP) (Ponting and Phillips, 1997; Symons etal., 1996). The EVH1 domains of Homer proteins from Drosophilia, rodentand human are conserved at a level of 80% identity (Xiao et al., 1998).Other than the IEG Homer 1a, all new forms of Homer encode an additionalC-terminal domain with predicted coiled-coil (CC) structure.

As the nomenclature suggests, Homer 1 gene encodes both the IEG form(Homer 1a) and splice forms that encode CC domains termed Homer 1b and1c. The 1b and 1c splice forms differ in their inclusion of anapproximately 10 amino acid sequence located between the EVH1 and CCdomains. (Homer family members that encode CC domains are also referredto as CC-Homers to distinguish them from Homer 1a, which lacks a CCdomain.) Similarly, Homer 2 encodes two CC-Homer splice forms termedHomer 2a and 2b, which also differ by a short internal sequence betweenEVH1 and CC domains. Homer 3 encodes a single form. The CC domains areless conserved than the EVH1 domain (˜40% identity between rat Homer 1,2 and 3) but they are able to specifically bind to themselves and toCC-domains of other Homer family members (Xiao et al., 1998). Homer CCdomains do not interact with other representative CC-domain proteins inGST pulldown assays, and a yeast 2-hybrid screen of brain cDNA with theCC-domain of Homer 1 identified multiple copies of Homer 1, Homer 2 andHomer 3, but not other CC domains (Xiao et al., 1998). As evidence thatHomer proteins can naturally self-multimerize, we demonstrated thatHomer 1b/Homer 3 heteromultimers co-immunoprecipitate from brain (Xiaoet al., 1998). These observations indicate that the Homer CC domainsmediate specific self-association.

In contrast to Homer 1a, all CC-containing Homer family members areconstitutively expressed in brain (Xiao et al., 1998). This wasconfirmed using both Northern blot and in situ hybridization assayswhich compared expression with Homer 1a in the same material. mRNA andprotein expression of Homer 1b/c, Homer 2 and Homer 3 are unchanged inhippocampus following a seizure while Homer 1a mRNA and protein areinduced at least 10 fold.

Antibodies were generated that specifically recognize each of theCC-Homers. Antibodies were raised against synthetic C-terminal peptidesequences. Because Homer 1b and 1c possess identical C-termini, theC-terminal antibodies recognize both splice forms. Similarly, C-terminalHomer 2 antibodies recognize both Homer 2a and 2b. Accordingly, whenusing these antibodies to detect Homer proteins, we refer to theimmunoreactivity as Homer 1b/c or Homer 2a/b. We used these antibodiesto determine that Homer 1b/c and 3 are enriched in a detergent resistantfraction of the postsynaptic density (PSD) (Xiao et al., 1998). Homer2a/b is also enriched in synaptic fractions, but is relatively moresoluble than Homer 1b/c and Homer 3. Like Homer 1a, each of theCC-Homers co-immunoprecipitates with group 1 mGluRs from brain (Xiao etal., 1998). Immunogold electron microscopy (EM) demonstrated that Homer1b/c and Homer 3 are ultrastructurally localized at the PSD (Xiao etal., 1998). These observations suggest that CC-Homer proteins functionas multivalent adapter complexes that bind mGluRs at postsynaptic sites.

Homer 1a Functions as a Natural Dominant Negative Protein.

The fact that Homer 1a lacks a CC domain suggested that it may functionas a natural dominant negative to disrupt cross-linking of CC-Homers. Inthis model, the EVH1 domain of Homer 1a can bind and compete for thesame target proteins as CC-Homers (such as mGluR5), but because Homer 1alacks the CC-domain, it cannot self-associate and cannot cross-link. Totest the dominant negative hypothesis, we generated a transgenic mousethat constitutively expressed Homer 1a in brain neurons under thecontrol of a modified Thy-1 promoter Aigner, 1995 #200. We confirmedtransgene expression in hippocampus, cerebellum and cortex in twoindependent lines (Xiao et al., 1998). The level of transgene expressionin the hippocampus was similar to natural Homer 1a expression induced bya seizure. In contract to the natural Homer 1a, however, the transgenewas constitutively expressed in the unstimulated mouse. A prediction ofthe dominant negative hypothesis is that the ability toco-immunoprecipitate mGluR with Homer 1b/c or Homer 3 antibodies shouldbe diminished in the transgenic mouse. As one of the controls for thisexperiment, we demonstrated by western blot that levels of expression ofmGluR1a, mGluR5 and Homer 1b/c, 2a/b, 3 were unchanged in the transgenicmouse brain. We then performed IP experiments and observed theanticipated result; the co-immunoprecipitation of mGluR5 with CC-Homersfrom hippocampus was reduced in the transgenic mouse (Xiao et al.,1998). Similar co-immunoprecipitations of mGluR1a with Homer 3 fromcerebellum was also reduced. As an additional control, we demonstratedthat the ability to co-immunoprecipitate Homer 1b/c with Homer 3 was notaltered in the transgenic mouse. This was the predicted result since theassociation between these proteins is mediated by their CC domains, andthis interaction is not altered by the Homer 1a EVH1 domain. Theseobservations support the hypothesis that Homer 1a functions as a naturaldominant negative to regulate CC-Homer-dependent cross-linking.

Homer Binds a Proline Rich Sequence that is ˜50 aa from the C-terminusof Group 1 mGluRs.

When we initially characterized the interaction between Homer andmGluR5, we anticipated that Homer might bind the free C-terminus. Thissurmise was based on the precedent of PDZ proteins such as PSD95 andGRIP, which bind the free C-terminus of NMDAR2 (Komau, 1995) and AMPAreceptors (Dong et al., 1997). Homer was noted to encode a GLGF sequencelike the PDZ domain. Additionally, in GST puildown assays that usedbrief washes, we noted a modest reduction of binding when the C-terminal4 or 10 aa were deleted from mGluR5 Brakeman, 1997 #99. (In retrospect,this modest reduction of binding may be due to Homer pulidown of Shankwhich does bind the free C-terminus of mGluR5, but appears to be loweraffinity than Homer-mGluR5 binding; see below.) However, with morestandard wash conditions, it became clear that the 4 and 10 aaC-terminal deletion mutants of mGluR5 continued to bind avidly to Homer.We continued the deletion strategy until we found that a 50 aaC-terminal deletion of mGluR5 destroyed binding to Homer. By contrast, a41 aa deletion of mGluR5 retained full binding activity. We noted thatthe intervening sequence was proline rich and shared sequence similaritywith the previously described SH3 ligand sequence [Yu, 1994 #166] Weprepared a series of point mutants based on the known structure-functionrelationship for SH3 ligands. Binding assays confirmed generalcharacteristics of SH3 ligand binding, but also demonstrated that thatthe Homer binding site is distinct in the positioning of critical aminoacids (Tu et al., 1998). A consensus for binding was determined to bePPXXFR (SEQ ID NO:13), consistent with the observation that mutation ofeither of the prolines or the phenylalanine, or a change in theirrelative position, interrupted binding. The arginine in the lastposition is preferred over other amino acids, but is not essential.Mutations were identically effective in interrupting binding to each ofthe Homer family members including Homer 1a, 1b/c, 2a/b, 3 and an EVH1only fragment (110 aa) of Homer 1. Thus, we conclude that theinteraction with mGluR5 is mediated by the Homer EVH1 domain.

Mutations of mGluR5 were initially tested in the context of a 250aaC-terminal fragment, but were also determined to have an identicaleffect on binding when placed in the full length mGluR5 protein (Tu etal., 1998). This exquisite sensitivity of Homer binding to changes insingle amino acid within the Homer-ligand site has been confirmed inother Homer-interacting proteins including mGluR1a (Tu et al., 1998),Shank (Tu et al., 1999) and I42 (see below). To further confirm that theinteraction was mediated by a direct interaction at the Homer-ligandsite (as opposed to a secondary allosteric effect on a remote bindingsite), we prepared synthetic 10 mer peptides with either the wild type,or F-to-R mutation, and demonstrated that the wild type peptide blockedbinding of mGluR1a or mGluR5 to each of the Homer family members (Tu etal., 1998). Approximately half of the binding was blocked at a peptideconcentration of 3.4 micromolar. By contrast, the F-to-R mutant peptidedid not alter binding at concentrations as high as 340 micromolar.

Homer Binds the IP3 Receptor.

Armed with a consensus sequence that predicted binding to Homer, wesearched the NCBI data base for other proteins that might bind Homer. AHomer-ligand site was identified in the IP3R, dynamin III, a human alphaadrenergic receptor and the ryanodine receptor (Tu et al., 1998). Eachof these interactions were determined to be consistent with the knowntopology of the candidate interacting protein, assuming that Homerproteins are cytosolic. We were able to confirm a biochemicalinteraction of Homer with the IP3R and dynamin III using GST pull downassays. More importantly, we demonstrated that the IP3Rco-immunoprecipitates with each of the Homer 1b/c, 2a/b and 3 fromdetergent extracts of cerebellum (Tu et al., 1998). Homer appears to beassociated with a substantial portion of IP3R in the cerebellum, since acocktail of the three Homer antibodies is able to specifically (comparedto a cocktail of preimmune serums) co-immunoprecipitate ˜50% of thetotal IP3R in detergent extracts (CHAPS).

CC-Homers Function to Link mGluR5 and IP3R in a Signaling Complex.

Based on the prior observations, we examined the hypothesis thatCC-Homers might cross-link mGluR and IP3R. This notion was appealing inthat the IP3R is part of the signaling network that is activated uponglutamate stimulation of mGluR1/5. Signaling complexes had previouslybeen described including; AKAP proteins which function as scaffolds forspecific kinases and their substrates Lester, 1997 #149, and theDrosophila protein InaD which couples the membrane light activatedchannel with its down stream effector enzyme, phospholipase C Tsunoda,1997 #147. Unlike these other examples of signaling complexes, however,Homer would need to form a bridge between receptors in two differentmembranes. Functional mGluRs are in the plasma membrane while the IP3Ris localized primarily to intracellular endoplasmic reticulum (ER). Insupport of the notion that ER and plasma membranes can come in closeapposition in neurons, we noted that Dr. Kristin Harris (Harvard)described the presence of smooth ER (SER, or spine apparatus) in thespines of hippocampal and cerebellar neurons (Tu et al., 1998).Remarkably, the SER forms close appositions with the plasma membranethat were uniquely localized to the lateral margin of the PSD. Thesesites are precisely where the group 1 mGluRs are localized (Baude etal., 1993; Lujan et al., 1997; Nusser et al., 1994). The IP3R is presentin spines of cerebellar Purkinje neurons where it is associated with thespine apparatus (Satoh et al., 1990). (Interestingly, in hippocampalneurons, the RYR is present in the spine apparatus while the IP3Rappears to be restricted to the dendritic shaft reviewed in (Narasimhanet al., 1998). Homer 1b/c and 3 are also enriched in the cytosol at thelateral margin of the PSD (Xiao et al., 1998). Thus, available anatomicevidence supported the notion that synaptic mGluRs come in closeapposition with SER-associated IP3Rs at sites that are enriched forCC-Homers.

As a first test of the hypothesis that CC-Homers cross-link mGluR andIP3R, we asked whether we could detect a trimolecular complex of mGluR,Homer and IP3R in brain. Indeed, IP3R antibody specificallyco-immunoprecipitated Homer and mGluR1a from cerebellum (Narasimhan etal., 1998). Since IP3Rs are not known to directly interact with mGluR1a,this result supported the hypothesis that Homer bridges these proteinsto form a trimolecular signaling complex. A further prediction of the“Homer hypothesis” is that Homer 1a should uncouple the putativemGluR-CC-Homer-IP3R complex. To test this, we monitored the effect ofHomer 1a expression on glutamate-induced intracellular calcium release.Plasmids expressing Homer 1a or Homer 1b were transfected along withgreen fluorescent protein (gene gun) and identified Purkinje neuronswere stimulated with quisqualate. A patch electrode containing the Ca2+detector Fura-2 was attached to the soma and a holding potential of −60mV was applied. Tetrodotoxin and picrotoxin were included in the bath toblock synaptic input and EDTA/MgCl₂ was included to assure that measuredCa2+ increases in the cell were generated from intracellular stores.Under these conditions, quisqualate-induced Ca2+ increases are due tomGluR1-evoked release from IP3R pools (Roche et al, J Biol Chem (1999)274:25953-259577). Expression of Homer 1b did not alter the induced Ca2+transient compared to cells transfected with an empty vector. Bycontrast, neurons transfected with Homer 1a showed a Ca2+ transient thatwas reduced in amplitude and delayed in time to peak (Tu et al., 1998).This result is consistent with the notion that the IP3 generated bymGluR1a activation of phospholipase C is less effective in releasingCa2+ from the IP3R pools in neurons expressing Homer 1a, and isanticipated if Homer 1a disrupts the physical linkage between mGluR1aand IP3R. Released IP3 must diffuse further, thereby resulting in alower effective concentration of IP3 at the receptor.

CC-Homers Alter Trafficking of mGluR1a/5 in Heterologous Cells.

We initiated studies to examine the effect of Homer on mGluR5expression. When wild type mGluR5 was expressed in heterologous cells(HEK293, COS or HeLa) the receptor reached the plasma membrane surfacewhere it was diffusely localized. This was also true when mGluR5 wasco-expressed with Homer 1a. However, we noted that co-expression ofmGluR5 with Homer 1b resulted in intracellular inclusions of mGluR5(Roche et al., 1999 supra). This effect of Homer 1b was dependent on theamount of transfected plasmid and was most obvious when equal amounts ofHomer 1b and mGluR5 plasmids were co-transfected. There was a trend forhigher level expression of mGluR5 when co-transfected with Homer 1b.When ratios of transfected plasmids were titrated so that total mGluR5expression was the same (comparing expression with or without Homer 1b),a substantial portion of the total mGluR5 was associated with theintracellular pool when co-expressed with Homer 1b. In these cells,relatively less reached the plasma membrane compared to mGluR expressedalone, or co-expressed with Homer 1a. We further noted that at earliertimes after transfection of Homer 1b and mGluR5, mGluR5 showed anenrichment in perinuclear organelles with a reticular pattern throughoutthe cell that resembled the ER. To assess the nature of theCC-Homer-dependent cellular accumulation, we compared the distributionof mGluR5 with the ER specific maker BIP B (Roche et al., 1999, supra).Staining with BIP antibodies revealed extensive ER present in bothtransfected and untransfected cells and co-localization with mGluR5. Wealso noted that the perinuclear organelles were not present withinnon-transfected cells and therefore appeared to be ER-derived structuresunique to cells overexpressing mGluR5 and Homer 1b. These observationssuggest that Homer 1b, but not Homer 1a, causes mGluR5 to be retained inthe ER.

As an additional assay for ER retention, we examined the status of thecarbohydrates present on mGluR5 in cells co-expressing Homer 1a or Homer1b. If Homer 1b caused mGluR5 to be retained within the ER, then mGluR5should contain immature, high mannose carbohydrates which are sensitiveto digestion with the enzyme Endoglycosidase H (Endo H). Alternatively,if mGluR5 had successfully traveled through the ER and cis Golgi, itwould possess mature, complex carbohydrates which would be Endo Hresistant. Mature carbohydrates would be anticipated if mGluR5 was onthe cell surface or if it was sequestered in a post-Golgi intracellularcompartment such as endosomes. We determined that mGluR5 is Endo Hresistant when expressed alone or with Homer 1a (Xiao et al., 1998).However, when expressed with H1b, mGluR5 is Endo H sensitive, consistentwith the hypothesis that expression of H1b leads to the retention ofgroup I mGluR in the ER.

The subcellular localization of the group II metabotropic glutamatereceptor mGluR2 was the same whether expressed alone or with H1b. Inaddition, we used a series of mGluR5 constructs containing pointmutations within the Homer binding site and found that mutations thatdisrupt mGluR5/Homer interactions in vitro also prevented ER retentionof mGluR5 co-expressed with H1b (Takei et al., 1994). mGluR5 P1125L,which does not bind to Homer in vitro (Tu et al., 1998), was notretained in the ER when co-expressed with H1b. In contrast, mGluR5S1126F, which does bind Homer in vitro, was ER retained whenco-expressed with H1b. Other point mutations in adjacent residues wereanalyzed and the results were consistent with in vitro binding studiessummarized in B (Ikeda et al., 1995), demonstrating that mGluR5 isretained within the ER by H1b only when its Homer binding site isintact.

While these experiments were performed in heterologous cells, we alsonoted enrichment of the group I metabotropic receptor mGluR1a in the ERof Purkinje cells (Kammermeier et al., submitted). Since Purkinjeneurons express particularly high levels of CC-Homers (Xiao et al.,1998), this suggests Homer proteins may naturally regulate receptortrafficking through the ER. In this model, Homer 1a would be permissivefor transfer through the ER Golgi system to insertion into thepostsynaptic membrane. The ability of CC-Homers to alter the spatialdistribution and metabolism of ER associated proteins may also impactthe IP3R. IP3Rs in Purkinje neurons are associated with dense stacks ofER (Satoh et al., 1990) and this stacking morphology has been shown tobe regulated by neural activity (Takei et al., 1994). Since asubstantial portion of IP3R in cerebellum is associated with CC-Homers,it is possible that the ability of CC-Homer to crosslink interactingproteins on two adjacent membranes plays a regulatory role in ERmorphology and function. Experiments in Aims 2 and 3 will examine thishypothesis.

Homer Modulates mGluR Coupling to Ion Channels.

Group 1 mGluRs modulate ionic currents by activating pertussistoxin-sensitive and -insensitive G proteins (Naisbitt et al., 1999).Modulation of Ca2+ currents by heterologously expressed group 1 mGluRsin superior cervical ganglion (SCG) neurons proceeds through multiplepathways involving both the a and βg-subunits of G proteins. We examinedthe effect of Homer on mGluR coupling to Ca2+ and M-type potassiumchannels in SCG neurons. CC-Homers, including 1b, 2b and 3 produced asimilar reduction of the effect of group 1 mGluRs (Kim et al., 1997;Naisbitt et al., 1999; Naisbitt et al., 1997; Takeuchi et al., 1997). Bycontrast, Homer 1a or an engineered short form of Homer 2 did not blockgroup 1 mGluR effects, but were able to partially reverse the effect ofthe CC-Homers.

Homer Interacts with Shank Suggesting a Role Synaptogenesis and NMDARFunction.

To gain further insight into the physiological function of Homer, wecharacterized a novel family of proteins that were identified based ontheir interaction with Homer 1a in a yeast 2-hybrid screen of a braincDNA library. These Homer-interacting proteins were determined to beidentical to the Shank family of PSD proteins that interact with GKAPand the PSD-95 complex (Tu et al., 1999). Shank proteins arespecifically enriched at excitatory synapses and co-localize with NMDAreceptors in primary neuronal cultures (Naisbitt et al., 1999). Shankproteins appear to be recruited to excitatory synapses by virtue oftheir interaction with GKAP, a synaptic protein that binds to theguanylate kinase domain of PSD-95 (Kim et al., 1997; Naisbitt et al.,1999; Naisbitt et al., 1997; Takeuchi et al., 1997). In addition to thePDZ domain which binds GKAP, Shank contains domains that mediateself-multimerization and interaction with cortactin (Golshani et al.,1998). Shank also directly interacts with Homer (Lujan et al., 1997).Homer and Shank proteins co-localize at the PSD of CA1 pyramidal neurons(Tu et al., 1999), and native Homer-Shank complexes were identified inbrain using GST pull down assays of Shank with GKAP (Otani and Connor,1998). Additionally, Homer and Shank co-immunoprecipitate from brain(Aniksztejn et al., 1991; Ben-Ari et al., 1992). These observationsindicate that Shank and Homer naturally associate in brain. Biochemicalstudies indicate that the Shank-Homer interaction is mediated by theEVH1 domain of Homer which binds to a single Homer-ligand site presentin the proline-rich domain of Shank proteins (Tu et al., 1999). Aquaternary complex of Homer/Shank/GKAP/PSD-95 is assembled inheterologous cells, with Homer and PSD-95 co-localizing in largeclusters (Berridge, 1998). Thus, Shank provides a molecular bridge thatlinks the NMDA receptor complex with Homer and its associated proteins.

The Homer-Shank interaction also produces clustering of group 1 mGluRs(Satoh et al., 1990; Villa et al., 1992). Clustering molecules havepreviously been identified for a variety of receptors and ion channels(Selig et al., 1995), but Shank-Homer are the first clustering proteinsfor group 1 mGluR. It is notable that the mechanism of clusteringinvolves a linkage of mGluRs with the previously defined NMDA receptorscaffold. Thus the Shank-Homer interaction could be relevant tosynaptogenesis, by docking mGluRs to a preestablished “core” of NMDAreceptors. In support of such a mechanism, functional NMDA receptorsappear to precede the emergence of metabotropic receptors in thehippocampus and cerebellum (Xiao et al., 1998). Homer proteins, inassociation with Shank, could function to localize and cluster themGluRs in proximity to NMDARs, and may contribute to the perisynapticlocalization of group 1 metabotropic receptors (Lujan et al., 1997).

By linking NMDA and mGluR signaling pathways, the Shank-Homerinteraction might also contribute to examples of glutamate receptorcrosstalk for which physical proximity of molecules may be important,such as activation of phospholipase C (Beneken et al., Neuron (2000)26:143-i54) or protein kinase C (Aniksztejn et al., 1991; Ben-Ari etal., 1992). Additionally, the Homer/Shank/GKAP/PSD-95 assembly maymediate physical association (and perhaps functional coupling) of theNMDAR with IP3R/RYR and intracellular Ca2+ stores. Consistent with sucha functional interaction, recent studies indicate that NMDAreceptor-dependent increases in spine Ca2+ may derive from intracellularstores by a mechanism of Ca2+-induced Ca2+ release (CICR) (Emptage etal., 1999) and reviewed by (Svoboda and Mainen, 1999). Both IP3R andryanodine receptor channels possess CICR properties (Berridge, 1998),and are similarly localized in dendrites and spines of specific neuronaltypes (Satoh et al., 1990; Villa et al., 1992). The physical proximityof glutamate receptors with calcium pools may underlie synergisticeffects of mGluRs on NMDA-dependent responses as reported in studies ofLTP (Bashir et al., 1993; Bortolotto et al., 1994) but see also ((Seliget al., 1995), and is consistent with the reduction of LTP in group ImGluR mutant mice (Prehoda et al., 1999).

The proposed model for Shank and Homer-dependent clustering requiresthat Homer be multivalent in order to cross-link Shank/GKAP/PSD95 toIP3R/RYRs and to mGluRs. This is achieved by multimerization ofconstitutively expressed CC-Homers (Xiao et al., 1998). In this context,the monovalent Homer 1a IEG product appears to function to uncoupleproteins that are linked via the constitutively expressed CC-Homermultimers, and thereby dynamically regulate the assembly of thispostsynaptic network. Cocaine-induced increases in Homer 1a may thusmodulate both mGluR and NMDA Ca2+ responses in spines.

Homer EVH1 Domain Crystal Structure.

To investigate the structural basis of interactions between EVH1 domainsand ligands, we determined the high-resolution crystal structure of theEVH1 domain from rat Homer 1. Methods of protein purification andcrystallization are described in our manuscript (Niebuhr et al., 1997;Tu et al., 1998). This structure revealed that the EVH1 module ishomologous to both the plextrin homology (PH) domain and thephosphotyrosine binding (PTB) domain.

At the same time we were working to solve the structure of Homer 1 EVH1,Dr. Wendel Lim's group (at UCSF) solved the structure of the relatedEVH1 protein termed Mena (20% identical to Homer EVH1 domain) (Prehodaet al., 1999). Comparison of the Mena and Homer coordinates confirmedthat these are related proteins despite the low degree of amino acididentity. The Mena crystal was solved with a 6mer peptide and identifieda putative ligand binding surface. Both of our groups determined thatco-crystals were not formed with longer synthetic peptides. One issuethat concerned us regarding the putative ligand-binding site on Mena wasthat the affinity of the 6mer used for Mena was 100 fold less than thatof a 10 mer (Prehoda et al., 1999). The measured affinity of the 6merwas ˜600 micromolar. Additionally, within the EVH1 family, Homer is oneof the most divergent members (Prehoda et al., 1999). One importantdifference between Mena and Homer EVH1 binding, is the orientation ofthe phenylalanine relative to the polyprolines. The optimal ligand forMena is FPPPP (SEO ID NO:47) while the consensus ligand for Homer isPPXXFR (SEQ ID NO:13). This may be important since the F is the singlemost critical side chain for the interaction when tested with largerpeptides for both EVH1 domains (Niebuhr et al., 1997; Tu et al., 1998).In the Mena structure, the F side chain is not placed in a clearhydrophobic pocket (the ring appears to coordinate an arginine) andsuperposition of the ligand coordinates in Homer EVH1 is even lessobviously stabilized.

To examine the predictive power of the Mena co-crystal for the ligandbinding activity of Homer EVH1, we tested a series of missense mutationsthat targeted sites anticipated to contact the prolines of the ligand(PPXXFR, SEQ ID NO:13) sequence. Based on the homology of the EVH1domain with the PTB domain, we also tested sites on Homer that would becritical if Homer mimicked the peptide binding surface of the PTBdomain. This PTB ligand site is remote from the putative Mena EVH1ligand site. Our mutation analysis also tested a series of mutantsselected based on the homology between Homer and WASP. Genetic data frompatients with Wiscott Aldrich syndrome defined a series of mutations inthe EVH1 domain that map to sites that are distinct from both the PTBand the putative Mena ligand sites. Our selection of the mutationalsubstitutions was based on the Homer EVH1 structure. Substituted aminoacids were selected to be sufficiently conservative as not to disruptthe primary structure.

A total of 30 missense mutants of the Homer EVH1 domain were expressedin HEK293 cells and assayed for binding to either mGluR1a or Shank3using GST pulldown assays. Surface-exposed mutations within the regionhomologous to the peptide binding site of PTB domains had no affect onpeptide binding. Similarly, mutations based on the WASP data were alsoineffective in disrupting binding. By contrast, certain of the mutantsbased the Mena ligand site did disrupt Homer EVH1 binding. Despiteambiguities involved with interpreting the effects of any singlemutation, the nature and distribution of the effects of site-directedmutations in the Homer EVH1 domain on Homer-ligand interactions stronglyimplicate the Mena ligand region as mediating natural ligand binding bythe Homer EVH1 domain.

One interesting finding from our analysis of mutant Homer EVH1 bindingis that certain mutations disrupt binding specifically to mGluR1a, butnot to Shank3 (and visa versa). One interpretation of this finding isthat there are determinants of binding in addition to the core PPXXFR(SEQ ID NO:13) motif. An important implication of this observation isthat differences in critical determinants of Homer binding to itsvarious targets may be exploited to develop pharmaceuticals that canselectively disrupt interactions with a particular target.

I42 Interacts with Homer.

I42 (SEQ ID NOS:17 and 18) encodes a novel protein that was firstidentified in a Y2H screen of brain cDNA with the Homer EVH1 domain.Current information indicates that I42 functions with Homer at theexcitatory synapse. We have generated I42 specific antisera and candemonstrate robust co-immunoprecipitation of I42 with Homer from brain.ImmunoEM analysis demonstrates that I42 is localized to the postsynapticdensity. The predicted domain structure of I42 indicates that it sharescertain properties with Shank including a N-terminal structural domain(a band 4.1 domain in I42), a single PDZ domain, and a central prolinerich domain with a single Homer-ligand site. Additionally, there is aC-terminal type 1 PDZ ligand motif. We have identified a relatedsequence in the data base (KIAA (SEQ ID NO:48) sequence has severalerrors with frame shifts) suggesting that I42 may represent a genefamily.

Current studies indicate a functional interaction of I42, Homer andmGluRs. We have performed a yeast 2-hybrid screen of the I42 PDZ domainand find it binds β-Pix (also termed Cool-1) (Allen et al., 1998). β-Pixis a guanine nucleotide exchange factor (GEF) for Rac1/CDC42. Thisinteraction appears robust using GST pulldown assays and we haverecently confirmed the interaction using co-immunoprecipitation assaysfrom brain. Biochemical assays indicate that the PDZ domain of I42 bindsits own C-terminus (may be intra or inter molecular). Based on theseobservations, I42 functions as a scaffold/cytoskeletal regulatoryprotein that responds to specific signals and may link between mGluRactivation and Rac-dependent cytoskeletal remodeling. This biochemicalassociation may play a role in mGluR trafficking or synaptic remodeling.An additional functional consequence of the Homer I42 interaction isindicated by the demonstrated association of β-Pix with p21 activatedkinase (Pak) (Tu et al., 1999). Paks are a family of kinase that cansignal both locally and more distally to the nucleus. A mutation of Pak3has recently been linked to mental retardation (Tu et al., 1998),confirming the importance of this regulated kinase to cognitivefunction. Accordingly, I42 appears to be part of a novel signalingpathway for the mGluRs that may be regulated by Homer proteins.

In preliminary studies, we observe that I42 co-immunoprecipitates withHomer from brain. Antibodies for I42 also co-immunoprecipitates mGluR1from brain. In parallel studies, we observed the interaction between I42and β-Pix ( ). These observations indicate the involvement of Homer inthe function of I42/β-Pix and identify another signaling pathway thatcan be manipulated by agents that modulate Homer binding function.

ii) Ultrastructural localization of I42/β-Pix/Pak at synapses: We haveperformed preliminary immunoEM with I42 Ab and observes that it isassociated with the PSD region. The methods and approach are identicalto our studies of Shank (Naisbitt et al., 1999). This observationindicates that I42 is enriched at the excitatory synapse together withHomer, Shank and glutamate receptors.

Ryanodine Receptor (RYR) and Homer.

The RYR encodes a potential Homer binding site near the N-terminus (Bhatet al., 1999) and using GST pulldown assays we observe that GSTHomerbinds to the relevant fragment of RYR1. Importantly, we havedemonstrated that the RYR co-immunoprecipitates with Homer fromdetergent extracts of skeletal muscle. The interaction between RYR andHomer is understood to be consistent with the function of Homer proteinsto regulate the coupling of membrane receptors with intracellularcalcium pools. Glutamate mediates an inhibitory postsynaptic potentialin dopamine neurons of the midbrain and this is mediated by mGluR1release of intracellular Ca2+ from RYR sensitive CICR pools (Bhat etal., 1999). RYR have recently been implicated as an important source ofNMDAR-induced calcium rise in the post synaptic spine (Emptage et al.,1999). Since Shank is part of the NMDA receptor signaling complex(Naisbitt et al., 1999) and binds Homer, it is compelling to evaluatethe possible interaction between RYR and Homer.

NMDA Receptor Type 2D (NR2D) and Homer.

Independent Y2H screens of adult cortex and cerebellum identifiedseveral clones of the NMDA receptor type 2D (NR2D). NR2D has not been asextensively studied as NR2B but is expressed in developing cerebellumand interneurons in the forebrain (Dunah et al., 1998; Goebel andPoosch, 1999). NMDAR that include the NR2DR have slower channelproperties (Cull-Candy et al., 1998; Okabe et al., 1998; Vicini et al.,1998). The C-terminus of NR2D is highly proline rich consistent with ourobservation that Homer binds a specific proline rich sequence. Thus, inthe case of NR2D, Homer proteins form a direct coupling to CICR pools.This direct coupling would contrast with NMDAR that include NR2B whichappear to couple to Homer indirectly via PSD95-GKAP-Shank (Naisbitt etal., 1999). In both cases, modification of Homer crosslinking activitywill alter the intracellular release of calcium due to glutamatereceptor activation. Because of the differences in the bindingproperties of the EVH1 domain of Homer to its different targets, it isanticipated that agents that specifically disrupt the linkage of NR2B orNR2D can be developed.

Mammalian InaD like Molecule Interaction with Homer.

We have identified two distinct novel members of a family of proteinswith similarity to the recently reported human InaD (Philipp andFlockerzi, 1997) and Drosophila Discs Lost DLT (Bhat et al., 1999).These proteins encode 5 and 4 PDZ domains, respectively, and a prolinerich region that is shared in all clones that is presumed to mediateinteraction with Homer. DLT has been demonstrated to be essential forestablishment of epithelial cell polarity and binds to the C-terminus ofNeurexin IV DLT (Bhat et al., 1999). We currently refer to our clones asrat InaD. In current studies, we observe that full length myc-taggedrInaD co-immunoprecipitates with Homer 2 from co-expressing HEK293cells.

I30 Interaction with Homer.

I30 is a novel member of the family of abl binding proteins. Relatedproteins function as adaptor proteins that regulate cell growthZiemnicka-Kotula, 1998 #392; Biesova, 1997 #393 and are hypothesized.I30 encodes a SH3 domain and a Homer binding site. Accordingly, Homer isanticipated to link this protein to other Homer-interacting proteinsincluding metabotropic glutamate receptors and IP3R. (See SEQ ID NOS:15, 16, 19 and 20).

Cdc42-Associated Tyrosine Kinase-2 (ACK-2) Interaction with Homer

ACK-2 is a non-receptor tyrosine kinase that is regulated by theRho-related GTP-binding protein Cdc42 Yang, 1999 #391. ACK-2 isactivated by signals that result from cell adhesion, by for exampleactivation of the integrin receptor. One cellular consequence of ACK-2activation is down stream activation of c-Jun kinase. Our observationthat ACK-2 interacts with Homer indicates that this signaling pathwaycan be linked to other membrane receptors by Homer, and identifiesanother signaling cascade that can be manipulated by agents that alterHomer crosslinking function.

Example 1 Identification and Sequencing of Homer Family Members

Low stringency screens of phage cDNA libraries and EST Database searcheswere performed to identify Homer family members. cDNA libraries werescreened using the rat Homer 1a coding region as a probe. Screens ofmouse and rat brain cDNA libraries identified two isoforms of Homer-1(Homer-1b and Homer-1c).

Searches of EST Databases identified a mouse EST sequence (ID#442801)which is about 73% homologous to a portion of 5′ coding region ofHomer-I cDNA sequence. Based on the EST used RT-PCR (Forward: 5′-GAC AGCAGA GCC AAC ACC GTG-3′; (SEQ ID NO:49); Reverse: 5′-GTC TGC AGC TCC ATCTCC CAC-3′; (SEQ ID NO:50)) to amplify the corresponding region fromvarious mouse tissues. The PCR products (˜330 bp) consisted of twodifferent sequences, one of which contains an additional insertion of 33bp. A mixture of these two cDNA fragments were used as probes to screenan adult mouse brain cDNA library. Out of 10⁶ clones screened, fiveclones hybridized well to the probe. Sequence analysis of these clonesindicated that they are five partial cDNA clones representing twoisoforms of a Homer-2 gene. These clones are identical to the isoformsamplified by RT-PCR. The 5′ region of Homer-2 was cloned using 5′-RACEtechnique. Total RNA from E14.5 mouse brain was reverse-transcribedusing the reverse primer described above. Another gene-specific primer(5′-CAC GGT GTT GGC TCT GCT GTC-3′; (SEQ ID NO:51)) was used in theamplification of the 5′ region of Homer-2. The sequence authenticity ofthe 5′ RACE clones was further confirmed by sequencing a partial mouseEST clone #441857.

A search of the EST Database allowed the identification of several humanEST's corresponding to mouse and rat Homer-1b, Homer-2a and 2b cDNAsequences. RT-PCR was used to clone the human Homer-1b and Homer 2a and2b coding regions. A 5′ degenerate primer (5′-ATG GG(A/G/C) GA(A/G)CA(A/G) CC(T/C/G) AT(T/C) TTC-3′; (SEQ ID NO:52)) was designed based onan amino-terminal seven residue amino acid sequence (MGEQPIF; (SEQ IDNO:53)) that is conserved among human EST clone#HCE003, mouse, rat, andDrosophila Homer homologue sequences. The 3′ primers (5′-GAG GGT AGC CAGTTC AGC CTC-3′; (SEQ ID NO:54)) for human Homer-1 and human Homer-2(5′-GTT GAT CTC ACT GCA TTG TTC-3′; (SEQ ID NO:55)) were made from thesequences of human EST clones #562862 and #HIBAB15 respectively. HumanHomer-1b and Homer-2a and 2b were amplified from new born human frontalcortex. The sequences of human Homer 1b, Homer 2a and Homer 2b werederived from sequencing several PCR clones and EST clones and are shownin SEQ ID NO's:3, 7 and 9.

Human and mouse Homer-3 were identified by searching EST Database, usingHomer-1 and Homer-2 sequences. Two full-length human Homer-3 clones wereidentified (Clone ID #284002 and #38753) and sequenced. Numerous mouseHomer-3 clones were found and one of them (Clone ID #1162828) containsan almost full-length coding region. Also identified were severalDrosophila EST sequences exhibiting significant homology at the aminoacid level to the N-terminal region of Homer family members. Thesequence presented in SEQ ID NO:9 is derived from Clone #LD3829.

Expression Constructs

Mammalian expression constructs were made by cloning cDNA into SalI andNotI sites of pRK5 (Genentech), so that the cDNA was fused in-frame toan N terminal c-Myc tag. GST-fusion constructs were made by cloningHomer cDNA into the SalI and NotI sites of pGEX4T-2 (Pharmacia). Thefull-length coding regions of mouse Homer-1b, rat Homer-1c, mouseHomer-2b and human Homer-3 were engineered with SalI and NotI sites atthe 5′ and 3′ ends by PCR using high fidelity DNA polymerase Pfu(Stratagene). Various truncations of Homer-1b/c and Homer-2b codingregions were made by PCR with specific Primers containing SalI and NotIsites. All the PCR-based constructs were sequenced to confirm thesequences and in-frame fusion.

The sequence of Homer 1a was used to screen cDNA libraries prepared fromrat and mouse brain for related gene products. Homer 1a sequence wasalso used to search GenBank data bases. Several related rodent and humansequences were identified.

cDNAs that are most closely related to Homer 1a appear to representalternative splice forms. This inference is based on nucleotide sequenceidentity of their 5′UTRs and the first 175 amino acids of the openreading frames (ORF). The presumptive novel splice variants, termedHomer 1b and 1c, are completely divergent from Homer 1a after residue175 of the ORF and they possess entirely distinct 3′UTRs. comparison atthe point of sequence divergence indicates that Homer 1a encodes aunique eleven amino acid carboxy terminus of the ORF and about 5 kb 3′UTR region. The unique eleven amino acid carboxy-terminal sequence ofHomer 1a does not possess a recognizable motif. In Homer 1b and 1c, anadditional 168 and 180 amino acids are present that are predicted topossess coiled-coil (CC) secondary structure (Lupas, Trends Biochem. Sci21:375 (1969)). While the 3′UTR sequence of Homer 1a includes multipleAUUUA repeats which are implicated in destabilizing mRNAs ofintermediate early genes (IEG) (Shaw and Kamen, Cell 46:659 (1986)), the3′UTR sequence of Homer 1b and 1c does not include this motif. The onlydifference between Homer 1b and 1c is the inclusion in Homer 1c of atwelve amino acid sequence insertion at residue 177, between theconserved amino-terminus and the CC domain. Thus, Homer 1b and 1c appearto be formed by a splicing event that substitutes a relatively long andunique carboxy-terminus of the ORF and shorter 3′UTR sequence that lacksthe characteristic IEG motif. Multiple independent isolates of rat andmouse Homer 1b and 1c were identified and sequenced to confirm theirnatural expression in brain.

Further searches identified cDNA sequences that appear to represent twoadditional Homer genes, termed Homer 2 and Homer 3. The sequences of twosplice forms of Homer 2 and one Homer 3 sequence is presented (SeeFigures section). The predicted size of the protein products and generaldomain structure are similar to Homer 1b and 1c. Like Homer 1b and 1c,each of the Homer 2 and Homer 3 proteins contain about 120 amino acidsat the amino-terminal that is highly similar to the amino-terminaldomain of Homer 1a. The degree of amino acid identity in these regionsis about 88% between Homer 1 and Homer 2 and about 86% between Homer 1and Homer 3. Many of the amino acid differences are conservative.

In contrast to the high degree of conservation in amino-terminal region,the carboxy-terminal regions of Homer 2 and 3 are only about 22%identical to Homer 1b, but like Homer 1b and 1c are predicted to possessa CC secondary structure. The CC domains of all Homer family membersexhibit significant homology (about 40-45% amino acid similarity) to theCC regions of myosin heavy chain (Strehler et al., J Mol Biol 190:291(1986)), kinesin heavy chain (Yang et al., Cell 56:879 (1989)) anddynactin (Gill et al., J Cell Biol 115:1639 (1991)). The distinct spliceforms of Homer 2, termed Homer 2a and Homer 2b, are differentiated by aneleven amino acid insertion at residue 131 in Homer 2b. Human Homer 1, 2and 3 are mapped to chromosomes 5, 15 and 19, respectively by the HumanGenome Project.

Drosophila Homer possess the basic domain structure of mammalian Homers.The amino-terminus is highly homologous to that of mammalian Homer andthe carboxy terminus is predicted to form a CC secondary structure.

Example 2 Generation and Characterization of Homer Antisera

Rabbit polyclonal antibodies were generated against synthetic peptidesderived from the unique carboxy termini of Homer 1b/c, Homer 2a/b andHomer 3. Synthetic carboxy-terminal peptides of Homer 1, 2 or 3 wereconjugated to thyroglobulin with glutaraldehyde and used to immunizerabbits according to a previously published protocol (Martin et al.,Neuron, 9:259 1992). Peptide sequences used are contained in Homer-1band 1c: IFELTELRDNLAKLLECS (SEQ ID NO:56); Homer-2a and 2b:GKIDDLHDFRRGLSKLGTDN (SEQ ID NO:57); and Homer-3: RLFELSELREGLARLAEAA(SEQ ID NO:58). Detergent (2% SDS) extracts from rat cortex,hippocampus, and cerebellum were separated on 8% SDS-PAGE gels andtransferred to nitrocellulose membranes. Blots was probed withpolyclonal anti-Homer sera. Specificity was tested by incubating theantiserum with 10 Tg/ml of relevant peptide at room temperature for 10 mprior to use. Rabbit polyclonal antiserum was also generated against thefull length GST-Homer 1a fusion protein, as described previously(Brakeman, et al., Cell 87:227 1997). This antiserum recognizes allHomer 1 isoforms.

Unpurified antibodies were tested for their sensitivity and specificityin detecting heterologously expressed, full length Homer proteins withamino-terminal c-myc tags. Each Homer protein was selectively detectedon Western blot by the appropriate Homer antibody in soluble extracts oftransfected HEK293 cells. The myc-tagged Homer proteins migrated with anapparent molecular mass of 50 kDa. There was no cross reactivity betweenantibodies for one Homer form and other family members.

Example 3 In Vitro Interaction of Homer Proteins with Cell-Surface mGluReceptors

To examine the interaction of Homer proteins with mGluR1 and mGluR5,HEK293 cells were transiently transfected (using calcium phosphate) withfull length mGluR1α and mGluR5 constructs in pRK5 (Brakeman et al.,1997). Cell lysates were made 24-48 h post-transfection. GST fusionproteins bound to glutathione agarose were prepared of Homer 1a, Homer1c, Homer 2b, Homer 3 and two amino terminal fragments of Homer 2according to the following procedure. GST fusion constructs wereprepared by polymerase chain reaction with specific primers thatincluded SalI and NotI sequences and subcloned into pGEX4T-2 vector(Pharmacia Biotech, Uppsala, Sweden). Constructs were confirmed bysequencing. GST-fusion proteins were expressed in BL21 bacterialstrains. Bacteria were harvested and lysed in PBS, 1% Triton X100, 2 mMphenylmethylsulfonyl fluoride (PMSF) and pelleted at 13,000 rpm (SorvallSS-34) at 4° C. for 5 m Proteins were purified by incubating 1 ml bedvolume glutathione-sepharose (GST) beads (Sigma USA) with bacterialsupernatant at 4° C. for 10 m, washing twice with PBS and PBS plus 1%Triton X-100. Protein was eluted with 10 mM glutathione and dialyzedagainst PBS at 4° C. Protein concentrations were measured by BCA(Pierce, Ill.). Cell lysates of the transfected cells were incubatedwith equivalent amounts of various Homer-GST fusion proteins at 4° C.for 2 h, washed with PBS and 1% Triton X-100. Proteins were eluted in 2%SDS sample buffer and separated on 8% or 2.5% SDS-PAGE gels and probedwith appropriate antibody.

It has been previously demonstrated that the amino-terminal 131 aminoacids of Homer 1a is sufficient to bind group I metabotropic glutamatereceptors (Brakeman et al., Nature 386: 284 (1997)). In view of the highdegree of sequence conservation in this region of Homer family members,the possibility that they would also bind group I receptors wasexamined. GST fusion proteins were prepared of Homer 1a, Homer 1c, Homer2b Homer 3 and two amino-terminal fragments of Homer 2. The fusionproteins were bound to glutathione agarose and assayed for binding tofull length mGluR5 or full length mGluR1a expressed in HEK293 cells.These studies show that mGluR5 bound GST Homer 1a. mGluR5 also bound toall full length Homer constructs and to a Homer 2 amino-terminalfragment of about 141 residues but not to GST alone. The relativebinding in the three assays were comparable for each of the three Homertypes. A Homer 2 deletion mutant that includes only the amino-terminal92 residues did not bind mGluR5. Similar binding of Homer proteins tomGluR1 was also observed.

Example 4 In Vivo Interaction of Homer Proteins with Cell-Surface mGluReceptors

To examine if Homer proteins are naturally associated with group Imetabotropic receptors in the brain, immunoprecipitation studies wereperformed. Rat or mouse brain tissues were sonicated (3×10 s) in PBS(˜200 mg/ml wet weight) containing 1% Triton-X100 with proteaseinhibitors and centrifuged for 10 m at 15,000 g. Three μl of antiserumdirected against Homer 1b, Homer 1c, Homer 2a, Homer 2b or Homer 3 wasadded to 60 μl of tissue extract and incubated for 1½ h at 4° C. andthen washed three times with PBS/Triton. Preimmune and peptide-blockedantisera were used as negative controls. Binding in tissue samples wasanalyzed by gel electrophoresis and western blot analysis. Proteins wereeluted in 2% SDS loading buffer. mGluR1α monoclonal antibody wasobtained from PharMingen (San Diego Calif.). Rabbit polyclonal mGluR5antibody was a gift from Dr. Richard Huganir, Johns Hopkins School ofMedicine.

Homer family members are naturally associated with group I metabotropicreceptors in brain. This analysis was performed using cerebellum sinceall three Homer family members are expressed in this tissue. Detergentextracts of whole adult rat cerebellum were incubated with antibodies toHomer 1b/c. Homer 2a/b or Homer 3 and immunopreciptates were blottedwith a mouse monoclonal antibody to mGluR1I. mGluR1Ico-immunoprecipitates with each of the antisera directed against Homerproteins. The predominate band after electrophoreses corresponded to themonomer form of mGluR1I (about 150 kDA) and other bands corresponding tomultimers of mGluR1I are also observed.

Example 5 In Vitro Interaction of Homer Proteins with IntracellularInositol Trisphosphate Receptors

To demonstrate that Homer proteins interact in vivo with inositoltrisphosphate receptors immunoprecipitation studies were performed usingbrain tissue. Rats or mice were sacrificed by decapitation and thecerebella were dissected immediately. Cerebella were sonicated in TEbuffer (50 mM Tris, 1 mM EDTA, pH 7.4) containing 1% CHAPS and proteaseinhibitor cocktail (˜00 mg wet weight/ml). The homogenate wascentrifuged at 90,000 rpm, 20 m, 4° C. in a TLA 100.3 rotor. 100 T1 ofthe cerebellar extract was used for each immunoprecipitation assay withthe following antibodies: 3 T1 of crude Homer 1, Homer 2 or Homer 3antibodies (Xiao et al., in press); 20 Tg of affinity purified inositoltrisphosphate antibody (gift from Alan Sharp). Antibodies and extractwere incubated for 30 m at 4° C., then 60 T1 of 1:1 protein A or proteinG (for goat antibody) sepharose slurry was added. Theantibody/extract/beads were incubated for an additional 90 m at 4° C.After washing 3×10 m in TE-CHAPS buffer, the proteins were eluted fromthe beads with 30 T1 of 4% SDS loading buffer and analyzed by SDS-PAGEand immunoblot.

Results from these studies showed that the inositol trisphosphatereceptor specifically co-precipitates with antisera directed againstHomer 1, Homer 2 and Homer 3.

Example 6 Calcium Mobilization is Decreased by Transient Expression ofHomer Protein Without a Coiled-Coil Domain

To demonstrate that Homer cross-links metabotropic glutamate receptorsand inositol trisphosphate receptors to provide or enhance a functionalsignaling complex, calcium mobilization was examined in cells transientexpressing truncated forms of Homer protein. The truncated Homer proteinused lacks the coiled-coil domain and is unable to form a bridge linkingthe mGluR at the cell surface with intracellular inositol trisphosphatereceptors. The truncated form of Homer protein resembled Homer 1a withthe exception of 11 residues at the carboxy-terminal. This form of Homerresults in enhanced expression of Homer protein as compared withtransfection of Homer 1a in heterologous cells. The Homer protein wasintroduced into Purkinje cells in primary cerebellar cultures andglutamate induced effects on calcium mobilization was measured.

Embryonic mouse cerebellar cultures were prepared and maintainedaccording to the method of Schilling et al. (Schilling et al., Neuron7:891 1991). At 4-5 DIV, cultures were transfected with plasmids codingfor E-GFP (Clontech) and either full-length Homer 1b or an IEG form ofHomer 1. The IEG form of Homer 1 was a 186 amino acid amino-terminalfragment of Homer 1b. Plasmids were purified by cesium banding. Threecombinations of the plasmids were transfected. Group I (control), 20 Tgof E-GFP and 40 Tg of pRK5 vector; group II, 20 Tg of E-GFP and 40 Tg ofpRK5 Homer 1 IEG; group III: 20 Tg of E-GFP and 40 Tg of pRK5 Homer 1b.Plasmid DNA was mixed with gold particles (0.6 micron), and coated ontoplastic tubing. DNA was then ballistically transfected into cellsaccording to the manufacturer's protocol (Helios Gene Gun System,BIO-RAD). After transfection, cultures were returned to the incubatorand maintained for an additional 2 days for a total of 7-8 DIV at thetime of use for imaging experiments.

Patch electrodes were attached to the somata of GFP-expressing Purkinjecells and a holding potential of −60 mV was applied. Micropressureelectrodes (1 Tm tip diameter) were filled with quisqualate (100 Tm inexternal saline) and were positioned ˜20 Tm away from large-caliberdendrites. Test pulses were delivered using positive pressure (6 psi, 1sec). Cells were bathed in a solution that contained (in mM) NaCl (140),KCl (5), EGTA (0.2), MgCl₂ (0.8), HEPES (10), glucose (10), tetrodotoxin(0.005), and picrotoxin (0.1), adjusted to pH 7.35 with NaOH, whichflowed at a rate of 0.5 ml/m The recording electrode contained CsCl(135), HEPES (10), fura-2 K₅ salt (0.2), and Na₂-ATP (4), adjusted to pH7.35 with C_(S)OH. Patch electrodes yielded a resistance of 3-5 MA whenmeasured with the internal and external salines described above.

Fura-2 ratio imaging of intracellular free Ca²+, was accomplished bymeasuring the background corrected fluorescence ratio at 340 and 380 nmexcitation using a cooled CCD camera system, as previously described(Linden et al., J Neurosci 15:5098 1995). Exposure times were 200 msecper single wavelength image. Experiments were conducted at roomtemperature. Enhanced GFP is weakly excited by illumination in the380-400 nm spectrum. Based upon the bandpass characteristics of our340HT15 and 380HT10 excitation filters and the absorption spectrum ofenhanced GFP (Clontech), we estimate that <1% of the signal at 340 nmexcitation and <5% of the signal at 380 nm excitation is contributed byGFP, even in those cells where the fura/GFP loading ratio is smallest.This could lead to a small (<5%) systemic underestimation of freecalcium concentration that should distribute randomly acrossexperimental groups.

Calcium mobilization in the absence of influx was measure by ratioimaging fura-2 in Purkinje cells bathed in Ca⁺²-free external saline andstimulated with a micropressure pulse of quisqualate, a metabotropicglutamate receptor agonist (Linden, Neuron 17:483 1996). The resultantCa⁺² transient is triggered by an mGluR and inositol trisphosphatepathway since it is completely blocked by either an mGluR antagonist((+)-MCPG, 500 TM in the bath) or a novel specific inositoltrisphosphate receptor-associated ion channel blocker, xestospongin C (1TM in the internal saline). Purkinje cells transfected with a truncatedform of Homer showed mGluR-evoked Ca⁺² responses with a decreasedamplitude (170±9 nM, mean±SEM, n=30 cells) and an increased latency(10.5±1.8 sec) as compared with cells transfected with Homer 1b (244±17nM, 4.2±0.9 sec, n=23) or an empty vector control (239±19 nM, 4.5±1.1sec, n=15). The decay phase of the Ca⁺² response appeared somewhatslower in neurons transfected with the truncated form. While the totalCa⁺² flux appeared similar in cells transfected with truncated andcomplete Homer proteins and in empty vector controls, the measurementcould not be made because the tail of the Ca⁺² response was abbreviateddue to the constraints of the image buffer capacity.

Example 7 Determination of the Crystal Structure of Homer Protein

The crystal structure of Homer protein and a Homer protein binding sitewere determined. Results of these experiments are presented in Table

(a) Protein Expression and Purification

Residues 1-120 of rat Homer 1a were expressed in Escherichia coli BL21cells as a C-terminal fusion to glutathione-S-transferase (GST-1aEVH) aspreviously described (Tu et al., Neuron 21:717 1998).Selenomethionine-substituted (SeMet) GST-1aEVH was prepared byexpression in the methionine auxotrophic strain B834 (DE3) (Novagen). 5mL of an overnight culture grown at 37° C. in LB media supplemented with100 Tg/mL ampicillin (Sigma) was added to 4L M9 minimal media (GibcoBRL) supplemented with 100 Tg/mL ampicillin, 0.05 mg/mL alanine,aspartic acid, glutamic acid, phenylalanine, glycine, histidine,isoleucine, lysine, asparagine, proline, glutamine, arginine, serine,threonine, valine, tryptophan, tyrosine, L-selenomethione, 1 μ/mLthiamine (Sigma), 2 mM MgSO₄, 1% glucose, 100 μM CaCl₂. Cells were grownto an A₆₀₀ of 0.5 at which time IPTG (Calbiochem) was added to a finalconcentration of 0.2 mM. Cells were grown for an additional 3 hours,harvested by centrifugation, and resuspended in 1× PBS/1% Triton.Pepstatin A and leupeptin (Boehringer-Mannheim) were added to a finalconcentration of 1 μg/mL, and PMSF (Life Technologies) was added to 0.5mM. Cells were lysed by sonication and centrifuged at 13,000 rpm in anSS-34 rotor to pellet cell debris. The cleared lysate was added to a 5mL glutathione-agarose (Sigma) column. The column was washed insuccession with twenty column volumes of 1× PBS/1% Triton, twenty columnvolumes of 1× PBS, and ten column volumes of cleavage buffer (50 mM Tris7.4, 150 mM NaCl, 2.5 mM CaCl₂, 50 mM θ-mercaptoethanol). All bufferswere degassed. A 50% slurry of glutathione-agarose beads loaded withfusion protein was incubated with 20 U of biotinylated Thrombin(Novagen) for 16 h at room temperature. The released cleavage product(1a-EVH) was collected, and the biotinylated Thrombin was removed withstreptavidin-agarose beads (Novagen). 1a-EVH was further purified bycation-exchange chromatography using a Resource S column(Amersham-Pharmacia).

(b) Crystallization and Data Collection

Crystals of native and SeMet protein were grown in hanging drops by themethod of vapor diffusion (Wlodawer et al., Proc Natl Acad Sci USA72:777 1975). 1T1 of a 9 mg/mL native or SeMet protein solution wasmixed with a 1:1 dilution of reservoir buffer (30% PEG 3350, 87 mMMgSO₄, 50 mM HEPES, pH 7.3) with distilled water and equilibrated over 1mL of reservoir buffer. All crystallization trials for the SeMet proteinwere set up under anaerobic conditions to minimize potential problemsdue to oxidation. Two different crystal forms were observed for both thenative and the SeMet protein. Crystals in the orthorhombic space groupP2₁2₁2₁, (unit cell dimensions a=33.79 Å, b=51.40 Å, c=66.30 Å)typically grew to a size of 0.5 mm×0.03 mm×0.03 mm. Crystals in thetrigonal space group P3₂21 (unit cell dimensions a=b=49.94 Å, c=80.91 Å)grew to a size of 0.4 mm×0.1 mm×0.1 mm. All data used for phasing andrefinement were collected from a single trigonal SeMet crystal soaked inmother liquor plus 10% (v/v) ethylene glycol for approximately threeminutes prior to flash freezing in a gaseous nitrogen stream at −180° C.X-ray diffraction data suitable for multiwavelength anomalous dispersion(MAD) phasing were collected at four wavelengths at or near the Seabsorption edge. These data were collected at beamline X4A of theNational Synchrotron Light Source at Brookhaven National Laboratoryusing an R-AXIS IV image plate detector. Nonoverlapping oscillations(2°) at φ and φ+180° were measured over a 90° rotation of the crystal,interleaving the four wavelengths. All data were processed and scaledusing the DENZO/SCALEPACK programs (Otwinowski and Minor, Meth Enzymol276:307 1997). Data collection statistics are shown in Table 1.

(c) Structure Solution and Refinement

The expected two selenium sites were determined and refined using theprogram SOLVE (Terwilliger and Berendzen, Acta Crystallogr D53:5711997;Terwilliger and Eisenberg, Acta Crystallogr A39:813 1983) and initial Sescattering factors from (Hall et al., Cell 91:85 1997). Values for therefined Se scattering factors as determined by SOLVE are shown inTable 1. The electron density maps calculated with the experimental MADphases as determined by SOLVE were improved by solvent flattening andhistogram matching using DM (Collaborative Computational Project, 1994).An initial model of residues 1-105 was built into 1.8 Å experimentalelectron density maps using the program O (Jones et al., ActaCrystallogr A47: 110 1991). After one round of simulated annealing withbulk solvent correction and positional and B-factor refinement using CNS(Brηnger et al., Acta Crystallogr D54:905 1998), residues 106-111 werebuilt into 2F₀-F_(c) maps. The model was refined against themaximum-likelihood target (Pannu and Read, Acta Crystallogr A52:6591996) using data to 1.7 Å Bragg spacing collected at 0.9879 Å. Eightrounds of model building and water addition alternated with B-factor andpositional refinement yielded the current model, which includes residues1-111 and 88 water molecules. No electron density was observed forresidues 112-120. This model has a crystallographic R value of 25.3% anda free R value of 28.4%. The solvent content is ca. 40.6%, with onemolecule per asymmetric unit. Fractional solvent accessibility for eachresidue was calculated in X-PLOR (Brünger, X-PLOR, Version 3.1: A systemfor X-ray crystallography and NMR (New Haven, Conn.: Yale Univ. 1992).

(d) Determination of Homer Site by Site Directed Mutagenesis

Point mutants of N-terminally myc-tagged, full-length Homer 1b and 1cand Homer 1 EVH1 were made using the QuikChange TM Site-DirectedMutagenesis Kit (Stratagene). Expression constructs were transientlytransfected into HEK293 cells using calcium phosphate methods. About24-48 h post-transfection, cell lysates were prepared in I× PBS/1%Triton X-100 (Sigma) and protease inhibitors. GST pull-down assays wereperformed by mixing 100 T1 of cell lysate with GST-mGluR5 or GST-Shank3(residues 1143-1408) (Tu et al., in press) bound to glutathione-agarose,incubating at 4° C. for 2 h, and washing with 1× PBS and 1× PBS/1%Triton X-100. Bound products were eluted with 100 T1 2× SDS loadingbuffer and detected by SDS-PAGE and immunoblot using anti-myc antibody9E10 (Invitrogen) and ECL reagents (Amersham).

Example 8 Homer Expression is Upregulated in Certain Brain Regions inResponse to Electrically Induced Seizures

Rat Homer 1a was cloned based on its rapid upregulation in hippocampalgranule cell neurons following electrically-induced seizure (MECS; seeBrakeman et al., Nature 386:284 1997) The expression of other members ofthe Homer family was examined in the brain following seizure.Radio-labeled riboprobes were prepared using unique sequences for Homer1a, Homer 1b, Homer 1c, Homer 2a, Homer 2b and Homer 3. Probes used didnot distinguish between the splice forms of Homer 1b and 1c or Homer 2and 2b.

(a) In situ Hybridization

Anti-sense and sense cRNA probes were generated from each mouse Homerplasmid by in vitro transcription in the presence of ³⁵SUTP, aspreviously described (Lyford et al., Neuron 14:433 1995). Probe forHomer-1a (Xiao, 1998; GenBank #AF093257) was derived from nucleotides1342 to 2140, for Homer 1b/c (Xiao, 1998; GenBank #AF093258) fromnucleotides 785 to 1396, for Homer-2a/b (Xiao, 1998; GenBank #AF093260submission) from nucleotides 486 to 1561, and for Homer-3 (GenBank#AF093261) from nucleotides 371 to 2123. Probe (about 10⁶ cpm in 75 T1hybridization buffer) was applied to each slide. Coverslipped slideswere then incubated in humidified chambers overnight at 56° C. Followingcompletion of wash steps, slides were air dried and exposed to KodakBiomax MR film for 2-3 days.

The anatomic distribution in unstimulated animals reveals thatexpression of Homer 1a is similar to the expression of Homer 1b andHomer 1c. High levels of expression of Homer 1a are observed in thehippocampus, striatum and cortex. In the cortex, there is laminarexpression with the highest levels in the superficial and deep layers.Expression of Homer 2a and 2b is enriched in the thalamus, olfactorybulb and principle neurons of the hippocampus in contrast to the cortexwhere low levels of expression of Homer 2a and 2b are observed. Homer 3is expressed primarily in the cerebellum and hippocampus.

In situ hybridization studies demonstrate the dramatic induction ofHomer 1a in response to MECS. In the hippocampus, induction ofexpression is estimated to be greater than 20-fold compared tohippocampus from unstimulated animals. MECS induced an increase in Homer1b and 1c expression of about 1.5 fold as determined by blot analysis.Expression of Homer 2 and Homer 3 is not altered in response to MECS.

Example 9 Formation of Multimeric Complexes of Homer Proteins

The CC secondary structure is implicated in protein-protein interactions(Lupas, 1996 supra). Therefore, the possibility that this domain mightconfer the ability to form homo- or hetero-multimers between Homerfamily members was examined. For examining the coiled-coil interactionof Homer family members, myc-tagged Homer-1c and Homer-2b weretransfected into HEK293 cells and cell extracts were made 2-3 dayspost-transfection. Cell lysates were treated as described above.

First, the ability of full length, bacterially-expressed GST fusionproteins of Homer to bind full length myc-tagged Homer proteinsexpressed in HEK293 cells was tested. myc Homer 1c bound Homer 1b, Homer2b, Homer 3, Homer 1b and Homer 2b carboxy-terminal CC domain, but notHomer 1a or Homer 2-amino-terminus. This is consistent with the notionthat the CC domain is important in the interaction, since Homer 1a andHomer 2-amino-terminus doe not encode the CC domain. To test thespecificity of the CC domain interactions, GST fusions of dynein IC-1aand dynein IC-2c were generated. The CC domains of these proteins showmodest sequence to Homer family CC domains and bind to the CC domain ofdynactin (Gill, 1991 supra). None of the myc-tagged Homer family membersbound to either dynein IC-1a or dynein IC-2c.

To determine whether Homer family members naturally form multimers inbrain, immunoprecipitates of cerebellum were examined. Extractsimmunoprecipitated with Homer 1b/c antibody contained Homer 3, whileextracts immunoprecipitated with Homer 3 contained Homer 1b/c. While itis possible that these co-immunoprecipitated Homer family members areassociated by means other than their CC domains, the fact theamino-terminus of Homer is monovalent and cannot form extendedconcatomers supports a model of multimerization mediated by the CCdomains. Homer 2 was not detected as a multimer with either Homer 1 orHomer 3 in these immunoprecipitation experiments.

Example 10 Homer Family Proteins are Enriched at Brain SynapticFractions and are Expressed in Certain Peripheral Tissues

The distribution and localization of Homer family proteins was examinedat the using immunochemical methods. Tissue extracts were assayed usingimmunoblot analysis and tissue localization was examined usingimmunohistochemistry at the light and ultrastructural levels.

(a) Immunoblot Analysis

Immunoblot staining of SDS (2%) extracts of various brain regions wereexamined to assess the distribution of Homer proteins in the brain.Homer 1b/c antibody detected a single band of about 47 kDa in cortex,hippocampus and cerebellum. These regions have similar levels ofexpression. The Homer 2a/b antibody detected a single major band in eachof cortex, hippocampus and cerebellum. Less intense, higher apparentmolecular mass bands were detected at about 60 and about 80 kDa. Homer 3immunoblots showed low level expression in cortex and hippocampus andintense staining of a single band in cerebellum (47 kDa).Immmunostaining was completely blocked by preincubating the antibodywith 10 Tg/ml of the relevant peptide antigen.

(b) Immunohistochemistry

For light microscopy, rats were deeply anesthetized with sevoflurane andperfused through the aorta with 250 ml of saline followed by 400 cc eachof 4% paraformaldehyde in 0.1% phosphate buffer (pH 6.5) and 4%paraformaldehyde in 0.1% phosphate buffer (pH 8.5). The rat was allowedto postfix for 1 hr. at room temperature and then prefused with 15%sucrose in 0.1% phosphate buffer (pH 7.4). The brain was removed andsectioned at 40 μm on a freezing sliding block microtome and collectedin PBS. Tissue was stained with an immunoperoxidase technique, asfollows. Brain sections were incubated in PBS containing 0.3% H₂0₂ and0.25% Triton X-100 for 30 m and then washed 3×5 m in PBS. Sections wereincubated in a buffer “PGT” containing 3% normal goat serum (ColoradoSerum Co.) and 0.25% Triton X-100 in PBS for 1 hr. and then transferredto the primary antiserum diluted 1:750 in the same PGT buffer. Sectionswere gently shaken for 48 h at 4° C., washed 4×5 m in PBS and thenincubated for 1 hr. at room temperature in a goat anti-rabbit IgGconjugated to horseradish peroxidase (Biosource International) diluted1:100 in PGT. Sections were washed 4×5 m in PBS and incubated for 6 m atroom temperature in 0.05% diaminobenzidine dihydrochloride (DAB:Sigma)and 0.01% H₂0₂ in 0.1 M phosphate buffer. Sections were washed in PBS,mounted onto gelatin chrome-alum subbed slides, dehydrated in a seriesof graded ethanol, cleared in xylene and coverslipped with DPX (BDHLimited).

Immunohistochemistry was performed to determine the cellularlocalization of Homer 1b/c and Homer 2a/b and Homer 3 in rat brain.Light microscopic examinations indicated that all three Homer proteinsare enriched in Purkinje neurons. Immunoreactivity is present in thecytoplasmic region of the soma and extends prominently into thedendritic arbor. The nucleus is not stained. Little or no staining isdetected in the contiguous granule cell layer. A similar lightmicroscopic pattern of cellular localization was detected for Homer 3.Homer 2 immunostaining in cerebellum also showed staining in Purkinjeneurons, but appeared technically less differentiated.

(c) Electron Microscopy

For EM, a postembedding immunogold method as described previously (Wang,et al., J Neurosci 18:1148 1998) was used and modified from the methodof (Matsubara, et al., J Neurosci 16:4457 1996). Briefly, maleSprague-Dawley rats were perfused with 4% paraformaldehyde plus 0.5%glutaraldehyde in 0.1 M phosphate buffer. Two hundred micrometerparasagittal sections of the rostral cerebellum (folia III-V) werecryoprotected in 30% glycerol and frozen in liquid propane in a Leica EMCPC. Frozen sections were immersed in 1.5% uranyl acetate in methanol at−90° C. in a Leica AFS freeze-substitution instrument, infiltrated withLowicryl HM 20 resin at −45° C., and polymerized with UV light. Thinsections were incubated in 0.1% sodium borohydride plus 50 mM glycine inTris-buffered saline/0.1% Triton X-100 (TBST), followed by 10% normalgoat serum (NGS) in TBST, primary antibody in 1% NGS/TBST, 10 nmimmunogold (Amersham) in 1% NGS/TBST plus 0.5% polyethylene glycol, andfinally staining in uranyl acetate and lead citrate. Primary antibodieswere used at dilutions of 1:500 for Homer 1b and 1:100-1:400 for Homer3.

Immunogold EM of Purkinje neurons of the cerebellum was performed todetermine whether Homer family proteins are associated with synapticstructures. Homer 1b/c showed striking localization to the region of thepostsynapic spine. Gold particles are densely concentrated in the regionof the postsynaptic density (PSD). A very similar distribution is notedfor Homer 3 immunoreactivity. It is noted that rather than beingconcentrated directed over the PSD or the contiguous plasma membrane,the majority of the gold particles appear to be present in the cytoplasmimmediately subjacent to these structures.

Peripheral Tissues Homer proteins are expressed in peripheral tissues.In detergent extracts of heart and kidney, a single band at 47 kDaimmunoreactive to Homer 1b and 1c is detected. In extracts of liver, acomplex of three bands ranging from about 44 to 47 kDa is detected. Inheart, liver, skeletal muscle and intestine, bands immunoreactive toHomer 2a and 2b are detected. Homer 3 immunoreactive bands are detectedin extracts of lung and thymus.

Subcellular Distribution To examine the subcellular distribution ofHomer proteins, a biochemical fractionation of rat forebrain wasperformed and fractions were analyzed by Western blotting with Homerantibodies. Fractions were blotted for mGluR5, BIP and synaptophysin tomonitor anticipated enrichment of fractions. Homer 1b/c, 2a/b and 3 werepresent in the crude nuclear pellet (P1), the medium spin crudesynaptosomal pellet (P2), and the high speed microsomal pellet (P3). BIPis a 78 kDa ER resident protein (Munro and Pelham, Cell 48:899 (1987)).and was enriched in both the P3 and the S3 fractions. While Homer 1b/cand Homer 3 were not abundant in the soluble (S3) fraction, Homer 2 wasenriched in the S3 fraction. The P2 fraction was subfractionated afterhypotonic lysis. The 25,000×g pellet (LP1), which is enriched in PSDs(Huttner et al., J Cell Biol 96:1374 (1983)), showed enriched presenceof mGluR5. The high speed pellet (165,000×g; LP2) showed the anticipatedenrichment in the synaptic vesicle protein synaptophysin (P38). Each ofthe Homer proteins was enriched in the LP1 fraction relative to LP2. thefinal soluble fraction (LS2) was uniquely enriched in Homer 2.

Example 11 Transgenic Mouse Model Demonstrates that Expression of Homer1a Selectively Blocks Binding of Homer 1b/c to mGluR5 in Vivo

N-terminal myc-tagged full-length Homer 1a ORF was cloned into theexpression vector pT2 (Gordon, et al., Cell 50:445 1987; Aigner, et al.,Cell 83:269 1995). Transgenic mice were generated at the University ofAlabama Transgenic Facility. Expression of the transgene protein wasassayed by western blot with rabbit polyclonal antisera that recognizesall Homer 1 isoforms (pan-Homer 1 antibody) and myc antibody.

Homer 1a is unique within the family of Homer related proteins in thatit is dynamically regulated and it lacks the CC domain. Accordingly, itwas hypothesized that the IEG would bind to group 1 metabotropicreceptors and disrupt the formation of multivalent complexes of Homerand mGluR. To examine this hypothesis, a transgenic mouse was generatedthat expresses Homer 1a under the control of a modified Thy-1 promoter(Gordon et al., 1987, supra), which drives neuron-specific expression inpostnatal brain (Aigner et al., 1995, supra). Transgenic mice expressedHomer 1a at high levels in cortex, hippocampus, cerebellum andthalamus/brainstem relative to levels in wild type litter mate controls.The pattern of Homer 1a transgene expression is consistent with thepreviously reported activity of this promoter (Gordon et al, 1987,supra). As expected, antibodies for both Homer 1b/c and Homer 2a/bco-immunoprecipitated mGluR5 from detergent extracts of wild typeforebrain. By contrast, Homer 1b/c antibody did not co-immunoprecipitatemGluR5 from transgenic mice. The effect of Homer 1a transgene expressionwas selective in that it did not disrupt the co-immunoprecipitation ofHomer 3 with Homer 1b/c. The latter observation is consistent with thenotion that the Homer 1b/c-Homer 3 interaction is mediated by the CCdomain and is predicted not to be altered by Homer 1a expression. Homer1a was not part of the complex co-immunoprecipitated with Homer 1b/c,consistent with the notion that the CC is necessary for association withthe complex. The effect of the Homer 1a transgene in blocking the invivo coupling of mGluR5 and Homer 1b/c was additionally selective inthat Homer 2 antibody co-immunoprecipitated mGluR5 similarly fromextracts of wild type and transgenic mice. Thus Homer 1a appears toselectively disrupt the interaction of Homer 1b/c with mGluR5 but notHomer 2 with mGluR5. Homer 3 is less highly expressed in forebrain thanHomer 1b/c or Homer 2a/b and co-immunoprecipitates of mGluR5 with Homer3 antibody were less clean. Accordingly, it could not be determined inthese experiments whether Homer 1a also competes with Homer 3. Identicalresults were obtained in tow independent mouse lines that express Homer1a transgene. The Homer 1a expressing transgenic mice have not beenbehaviorally characterized but appear normal in size and gross motoractivity.

Example 12 Yeast Two-Hybrid Screen

To examine the physiological functions of Homer, a novel family ofproteins was identified based on its ability to interact with Homerfamily proteins in a yeast two-hybrid screen of a brain cDNA library.Homer 1a was subcloned into pPC97 (Chevray and Nathans, Proc. Natl.Acad. Sci. U.S.A., 89:5789 (1992)) and used to screen a random primedcDNA library prepared from seizure-stimulated rat hippocampus and cortexcloned in pPC86 (Chevray and Nathans, 1992, id.) as described previously(Brakeman et al., Nature, 386:284 (1997)). The same library wasrescreened using the PDZ domain of Shank 3 (amino acid residues 559-673)cloned into pPC86. The Shank 3 PDZ domain was also tested forinteraction with mGluR constructs in pPC86. mGluR5 constructs included awild type C-terminal 241 amino acid fragment and a four amino acidcarboxy-terminal deletion of the same fragment.

Using Homer as “bait” in a yeast two-hybrid screen of a rat cortex andhippocampus cDNA library, multiple cDNA isolates of two novel genes wereobtained. Sequencing and full length cloning identified these asdistinct members of a gene family, termed Shank 1 and 3 (Naisbitt etal., Neuron (1999) 23:569-82). Shank family proteins are closely relatedto a previously described protein, termed Cortactin Binding protein(CortBP-1; Du et al., Mol. Cell. Biol., 18:5838 (1998)).

Example 13 Interactions Between Homer Proteins and Shank Proteins inVitro and in Vivo

To characterize the interaction between Homer proteins and Shankproteins, the Shank cDNAs isolated from the yeast two-hybrid screen(Example 10)) were expressed in HEK293 for GST pulldown assays withGST-Homer 1a. The interaction between Homer and Shank proteins wasfurther characterized by co-immunoprecipitation assays.

(a) Expression Constructs

Shank expression constructs were prepared as described (Naisbitt et al.,in press). Site directed point mutants of Shank were generated usingQuik Change (Stratagene). GST fusion constructs were prepared bypolymerase chain reaction (PCR) using Pfu Polymerase (Stratagene) withspecific primers that included SalI and NotI sequences. After digestionwith SalI/NotI, PCR products were subcloned into pGEX4T-2 vector(Pharmacia Biotech, Uppsala, Sweden) or N-myc-tagged pRK 5 vector(modified from Genentech). All constructs were confirmed by sequencing.GST-fusion proteins were expressed in BL21 E. coli strains (GIBCO, BRL).Bacteria were harvested and lysed in PBS, 1% Triton X-100, 2 mMphenylmethylsulfonyl fluoride (PMSF) and pelleted at 13,000 rpm (SorvallSS-34) at 4° C. for 5 m. Proteins were purified by incubating 1 ml bedvolume glutathione-sepharose beads (Sigma) with bacterial supernatant at4° C. for 10 m. and washed twice with PBS and PBS plus 1% Triton X-100.Bound proteins were eluted with 10 mM glutathione and dialyzed againstPBS at 4° C. Protein concentrations were measured by BCA (Pierce, Ill.).mGluR5 constructs and mutants are described in Tu et al., Neuron 21:717(1998).

(b) GST pulldown and Co-immunoprecipitation Assays

Expression constructs were transiently transfected into HEK293 cellsusing the calcium phosphate method. Cells were lysed 24-48 hpost-transfection with PBS plus 1% Triton X-100. GST pull down assayswere performed by mixing 100 T1 cell lysates with beads charged with GSTfusion proteins (1-3 Tg/50 T1 bed vol.) at 4° C. for 2 h followed bywashing once with PBS, once with PBS plus 1% Triton X-100. Boundproteins were eluted with 100 T1 2× SDS loading buffer and detected bySDS-PAGE and immunoblotting using ECL reagents (Amersham). GST pull downassays of mGluR1a and mGluR5 from brain lysates were performed bysonicating rat cerebellum or cortex in 50 mM Tris, 1 mM EDTA, 1% CHAPS(Sigma), 0.5% deoxycholic acid (Sigma) and proteinase inhibitors withGST-proteins and these tissue extracts were then processed as above. Forimmunoprecipitation from COS7 cells, transfected cells were extracted inRIPA (see Naisbitt et al., 1999, supra). Soluble extracts wereprecipitated with 2 Tg control non-immune IgG, Myc or Shank 1 (56/e)antibodies (Naisbitt et al., 1999, supra).

Extracts of forebrain crude synaptosomes for immunoprecipitation wereprepared using deoxycholic acid as described previously (Dunah et al.,Mol. Pharmacol. 53429 (1998)). Forebrain P2 fraction was extracted in 1%deoxycholic acid, dialyzed over night into 0.1% Triton X-100, 50 mMTris, pH 7.4. Concurrently, 5 Tg of each antibody was pre-incubatedovernight with 10 T1 bed volume protein A-sepharose. Aftercentrifugation at 100,000 g for 1 h, 50 Tg of extract was incubated withantibody-protein A in 100 T1 0.1% Triton X-100, 50 mM Tris, pH 7.4 for 2h at 4° C. Pellets were washed 4 times with 1 ml incubation buffer, andbound proteins were analyzed by immunoblotting.

Antibodies Shank antibodies were raised in rabbits immunized withGST-fusions of Shank 3 residues 1379-1740 and 1379-1675 (Covance,Denver, Pa.). Similar bands were seen on rat brain immunoblots with bothantisera. GKAP, PSD 95 and Shank 1 (56/e) antibodies are described in(Naisbitt et al., 1999, supra). Homer antibodies are described above.Anti-mGluR 1a monoclonal antibody is from Pharmingen and rabbitpolyclonal mGluR5 antiserum was obtained from Dr Richard Huganir (JohnsHopkins University).

Shank cDNAs derived from the yeast two-hybrid screen were expressed inHEK293 cells for GST pulldown assays with GST-Homer 1a. Each of theShank polypeptides specifically bound Homer 1a. Based on the findingthat the Homer EVH1 domain binds a specific proline-rich motif threepotential Homer binding sites (or Homer “ligands”) that are conserved inShank 1, 2, 3 and CortBP-1 were identified.(Naisbitt et al., 1999,supra). To define the Homer binding site on Shank family proteins, threedeletion fragments of Shank 3 that included, respectively, amino acidresidues 559-908, amino acid residues 1143-1408, and amino acid residues1379-1740 were testing for their ability to bind to Homer 1b, Homer 1c,Homer 2 and Homer 3 in GST puildown assays. Similar binding specificitywas detected with each of the Homer proteins. Only Shank3 fragment1143-1408 bound to Homer. This region contains the amino acid sequencethat most closely resembles the Homer ligand peptide consensus(LVPPPEEFAN; residues 1307-1316; SEQ ID NO:59). A similar sequence ispresent in Shank1 (PLPPPLEFSN 1563-1572; SEQ ID NO:60: see Naisbitt etal., 1999, supra). CortBP possesses two similar sites; (PLPPPLEFAN;residues 813-822; SEQ ID NO:61) and (FLPPPESFDA residues 878-887; SEQ IDNO:62). Fragments of Shank3 containing amino acid residues locatednearer the amino-terminal of the protein such as Shank 3 fragment559-908 (which includes the PDZ domain and the first proline-rich motif)did not bind to Homer, but did bind to GKAP (Naisbitt et al., 1999,supra). Similarly, Shank3 fragment 1379-1740, which includes thecarboxy-terminal proline-rich sequence and the SAM domain, did not bindto Homer, though it is capable of binding itself and cortactin (Naisbittet al., 1999, supra). These studies identify the Homer binding site asbeing distinct from either the PDZ domain that binds GKAP, or theproline-rich binding site that binds cortactin and which is locatednearer to the carboxy-terminal (Naisbitt et al., 1999, supra).

To confirm the site of Homer interaction, site directed point mutants ofthe putative Homer ligand in Shank3 were assessed for their ability tobind to GST-Homer 1c. Full length wild type Shank 3, Shank3(P1311L), andShank3(F1314C) were expressed in HEK293 cells and assayed for binding toGST-Homer 1c. Compared to wild type Shank 3, both point mutants showeddramatically reduced binding to Homer. These experiments provide furtherconfirmation that the Homer ligand in Shank3 is the principle site ofinteraction.

It has been previously demonstrated that amino acids 1-110 of the HomerEVH1 domain are necessary and sufficient for binding to Homer ligands(Brakeman et al., 1997, supra; Tu et al., 1998, supra). To confirm thatthe EVH1 domain of Homer mediates interactions with Shank, a series ofpoint mutants of the Homer 1 EVH1 domain were generated. Mutations thatdisrupted binding to mGluR5 disrupted binding to Shank 3 in an identicalmanner, indicating Homer binds both proteins via a similarEVH1-dependent mechanism (Beneken et al., 2000, supra).

To confirm the interaction between Homer and Shank in a mammalian cellcontext, co-immunoprecipitation experiments were performed inheterologous cells. COS7 cell were transfected with Myc tagged-Homer 1b,Shank 1, or Shank 1 plus myc-Homer 1b. Detergent extracts of cells weresubjected to immunoprecipitation and blotted with myc, shank, or control(non-immune IgG) antibodies. Homer 1b was used in these experimentsbecause it expresses more efficiently in mammalian cells than Homer 1a.There is co-immunoprecipitation of Homer with Shank antibody and ofShank with myc antibody only from cells expressing both Shank andmyc-Homer 1b.

To demonstrate the in vivo relevance of the Homer-Shank interaction,co-immunoprecipitation experiments were performed using detergentextracts of rat brain. Detergent extracts of rat forebrain fractionswere immunoprecipitated with Shank and control (non-immune) antisera.Immunoprecipitates were blotted for Homer, Shank and GRIP antibodies.Antibodies raised against a fusion protein of Shank 1 immunoprecipitatedHomer 1b and 1c proteins as well as Shank from rat forebrain. GRIP wasnot co-immunoprecipitated with Shank and neither Shank or Homer wereprecipitated by non-immune IgGs. Furthermore, another Shank antibody,generated against Shank 3 fragment 1379-1675, co-immunoprecipitatedHomer 1b and 1c extracted from both cerebellum and cortex.

Example 14 Homer and Shank Mediate Clustering of Cell-Surface Receptors

Shank proteins may link Homer proteins with components of a cell-surfaceclustering complex, such as the NMDA clustering complex.

COS7 cells were transfected using the Lipofectamine method (GIBCO-BRL)on poly-lysine coated coverslips for clustering experiments, asdescribed in Naisbitt et al. ([in press] 1999, supra) and Kim et al.(Neuron 17:103 1996). Primary antibodies were used as follows: GKAPC9589, 1 Tg/ml (Naisbitt et al., 1999, supra); Shank 56/e 0.5 Tg/ml(Naisbitt et al., 1999, supra), PSD-95, 1:1000 diluted guinea pig serum(Kim et al., Neuron 378:85 1995). Cy3 and (fluoroscein isothiocyanateconjugate (FITC)-conjugated secondary antibodies (JacksonImmunoresearch) were used at dilutions of 1:500 and 1:100 respectively.

Yeast two-hybrid screens were performed as described in Example 10.

A yeast two-hybrid screen of the same rat brain cDNA library wasperformed using the PDZ domain of Shank3 as bait. From this screen, twoidentical clones of the carboxy-terminus of GKAP-3/SAPAP3 were isolated.In a reciprocal screen, Naisbitt et al., 1999, supra) isolated multipleclones of Shank1, 2 and 3 using GKAP as bait. This result providesindependent confirmation of the specificity of the interaction betweenthe Shank and GKAP/SAPAP families of proteins.

The cDNA from the yeast two-hybrid screen encoding the carboxy-terminal347 amino acids of GKAP-3 was expressed with an amino-terminal myc tagin HEK293 cells and tested for binding to GST fusion constructs ofShank3 and other PDZ containing proteins. The GST fusion of Shank3fragments containing just the PDZ domain (residues 559-673) wassufficient to bind GKAP3, while a Shank3 construct lacking the PDZdomain (residues 665-908) failed to bind. Additionally, PDZ domains ofGRIP and SAP102 failed to pull down GKAP3, demonstrating the specificityof the Shank-GKAP interaction.

The above findings suggest that Homer, Shank and GKAP may assemble intoa ternary complex. To explore this further, GST pull-down assays wereperformed using rat brain extracts. The carboxy-terminal 76 amino acidsof GKAP 1a, containing the Shank PDZ-binding sequence -QTRL, was fusedto GST GST-GKAP(carboxy-terminal). GST-GKAP(carboxy-terminal)specifically pulled down both Shank and Homer 1b and 1c, but not GKAP1or several other proteins (Naisbitt et al., 1999, supra). Since GKAPbinds directly to Shank but not to Homer (Naisbitt et al., 1999, supra),the results suggest that the GKAP pulldown of Homer is mediated byShank. These findings corroborate the co-immunoprecipitation experimentsof Shank and Homer from brain extracts and confirm that Homer isassociated with Shank in a native complex.

Since Shank proteins may link Homer proteins with components of the NMDAclustering complex, co-clustering of these proteins in transfected COScells was assessed. In cells co-expressing Homer 1b and PSD-95, bothproteins showed a diffuse distribution in the cytoplasm. This is notsurprising, since Homer and PSD-95 do not interact directly. When cellswere transfected with Shank1 and GKAP in addition to Homer and PSD-95,Homer and PSD-95 redistributed into plaque-like clusters in which bothproteins were exactly co-localized. By contrast, co-clustering of Homerand PSD-95 was not observed following co-transfection of Homer andPSD-95 with either Shank1 or GKAP alone. Thus, Homer and PSD-95co-cluster only upon co-expression of Shank and GKAP. Therefore, Shankand GKAP may mediate the formation of a quaternary protein complexcontaining PSD-95 and Homer (see also Naisbitt et al., 1999, supra).Other types of macromolecular complexes may also form when Homer andShank proteins interact. Cells expressing Homer 1b and Shank 1 (withoutGKAP or PSD-95) exhibited a redistribution of Homer 1b into a reticularfilamentous pattern, as well as into clusters; in both kinds ofstructures Shank and Homer immunoreactivities were co-localized. Thesefindings provide further evidence for an interaction between Homer andShank, and suggest that Homer 1b and Shank can co-assemble into higherorder macrocomplexes. This result is consistent with the biochemicalproperties of Shank that include its ability to self-multimerize andbind cortactin (Naisbitt et al., 1999, supra). Since Shank, GKAP, andPSD-95 are components of NMDA receptor-associated complex (Naisbitt etal., 1999, supra), the identification of Homer as a Shank-bindingprotein invokes a molecular link between the NMDA receptor complex andHomer-associated synaptic proteins such as mGluR1a and 5 and theinositol trisphosphate receptor.

Group 1 Metabotropic Receptors Based on the observations in heterologouscells that Shank clusters with Homer 1b and that Shank together withGKAP can mediate the co-clustering of Homer and PSD-95 Shank may mediateclustering of group 1 metabotropic glutamate receptors (mGluRs).Co-expression of Shank1 and mGluR5 in COS cells did not result inobvious clustering of either protein. Similarly, Homer and mGluR5 do notform co-clusters. Co-expression of the three proteins Homer, Shank 1,and mGluR5, however, resulted in conspicuous co-clustering of mGluR5with Shank 1. Clustering of mGluR5 in these triply transfected cells wasdependent on the ability of Homer to bind the receptor since a pointmutant of mGluR5 that does not interact with Homer failed to co-clusterwith Shank. Thus, both Homer and Shank are required to mediate theclustering of mGluR5.

Example 15 The Shank 3 PDZ Domain Binds the Carboxy-Terminus of Group 1Metabotropic Receptors Directly at a Site Distinct from the HomerBinding Site

The Shank PDZ domain shows selective binding to the GKAPcarboxy-terminus (Naisbitt et al., 1999, supra). The carboxy-terminalsequence of GKAP (-QTRL) finds similarities with that of the group 1mGluRs (mGluR1a -SSSL; mGluR5-SSTL) and therefore it was determinedwhether the PDZ domain of Shank can directly bind the carboxy-terminusof group 1 mGluRs. GST-pulldown assays were performed using extractsfrom heterologous cells expressing a recombinant mGluR5 carboxy-terminal241 amino acid peptide. The mGluR5 carboxy-terminal tail bound twopartially overlapping constructs of Shank 3 that included the PDZ domain(559-908; and 559-673), but not a construct from which the PDZ domainwas deleted (amino acids 665-908). Binding of mGluR to the Shank3 PDZdomain was qualitatively similar to mGluR5 binding to Homer 1c and Homer2. Negative controls included absence of binding of mGluR to SAP102PDZ1-3 and GRIP PDZ 4-6. Furthermore, a deletion mutant of the mGluR5polypeptide that lacked the carboxy-terminal four amino acids failed tobind to the PDZ domain of Shank3. Identical interactions between ShankPDZ and mGluR5 C-terminal tail were detected in a yeast two-hybridanalysis. These studies indicate that the PDZ domain of Shank 3 can bindthe carboxy-terminus of group 1 metabotropic receptors via aPDZ-mediated interaction with the carboxy-terminal sequence—S S/T L.

To confirm that Shank3 PDZ domain can bind full length native mGluRs,GST pull down assays were performed with detergent extracts of forebrainor cerebellum. The PDZ domain of Shank 3 bound specifically to mGluR1aand mGluR5 from cerebellum and forebrain, respectively. (Cerebellumpredominantly expresses mGluR1, while forebrain expresses predominantlymGluR5.) While it is possible that the Shank3 PDZ pulldown of mGluRsfrom brain extracts is indirect, via Shank PDZ pulling down aGKAP-Shank-Homer-mGluR complex, this extended complex is unlikely giventhe more modest ability of GST-GKAP to pull down Homer.

These studies suggest that Shank may interact with the cytoplasmic tailof mGluR1a/5 both directly, via its PDZ domain, and indirectly, viaHomer. The inability of Shank 1 to cluster mGluR5 in the absence ofHomer indicates that the direct PDZ-dependent Shank-mGluR interaction iscontingent upon a co-incident Homer interaction. Both modes ofinteraction with mGluR may be involved in mGluR clustering by Shank andcontribute to physiological regulation.

Example 16 Shank and Homer Co-Localization at Specific Post SynapticDensities

Immuno Electron Microscopy A postembedding immunogold method (Petraliaet al., Nature Neurosci 2:31 1999; Zhao et al., J Neurosci 18:5517 1998)was used. Male Sprague-Dawley rats was perfused with 4% paraformaldehydeplus 0.5% glutaraldehyde in 0.1 M phosphate buffer (PBS). Parasagittalsections (250 Tm) of the hippocampus were cryoprotected in 30% glyceroland frozen in liquid propane in a Leica EM CPC. Frozen sections wereimmersed in 1.5% uranyl acetate in methanol at −90° C. in a Leica AFSfreeze-substitution instrument, infiltrated with Lowicryl HM 20 resin at−45° C., and polymerized with UV light. Thin sections were incubated in0.1% sodium borohydride plus 50 mM glycine in Tris-buffered saline/0.1%Triton X-100 (TBST), followed by incubations in 10% normal goat serum(NGS) in TBST, primary antibody in 1% NGS/TBST, 10 nm immunogold(Amersham) in 1% NGS/TBST plus 0.5% polyethylene glycol, and finallystaining with uranyl acetate and lead citrate. For double labeling, thefirst primary antibody (e.g., Shank; Shank3 1379-1675 antigen) andcorresponding immunogold-conjugated antibody (10 nm gold) were applied,sections were exposed to paraformaldehyde vapors at 80° C. for one hour,and the second primary (Homer 1b and 1c) and secondary (20 nm gold; TedPella/BBI International) antibodies were applied the following day.Controls (showing little or no gold labeling) included absence of theprimary antibody for single labeling and absence of the second primaryantibody for double labeling. Primary antibodies were used at dilutionsof 1:100-1:300 for Shank and 1:400 for Homer 1b and 1c.

An antibody generated against a carboxy-terminal region of Shank 3(amino acids 1379-1675) was used to examine the ultrastructualdistribution of the Shank proteins in brain. This antibody recognizesmultiple bands on brain immunoblots, including major bands of ˜160-180kD and ˜210 kD in forebrain and cerebellum, similar to those seen withother Shank antibodies (see Naisbitt et al., 1999, supra). The differentsize bands presumably derive from the multiple Shank genes and splicevariants. All Shank immunoreactivity is blocked by incubation of theShank antibody with the Shank fusion protein antigen.

Immunogold electron microscopy revealed intense Shank immunoreactivityat the PSD of CA1 pyramidal neurons. Gold particles were distributedover the entire region of the PSD. In the same preparations, Homer 1b/1c was found to co-distribute with Shank. In all profiles withimmunostaining for both Shank and Homer, gold particles were presentover the PSD but also extended into the region subjacent to the PSD.This distribution is similar to the distribution of NMDA receptorsassociated with the postsynaptic membrane (Petralia et al., 1999, supra)and distinct from the distribution of mGluR5 which are most prevalent inthe perisynaptic membrane region just outside the PSD (Lujan et al., EurJ Neurosci 8:1488 1996). This spatial localization is consistent withthe idea that Shank 3 and Homer interact with components of both theNMDA receptor and metabotropic receptor signaling complexes.

This family of proteins that interact with Homer are identical to theShank family of postsynaptic density (PSD) proteins that interact withGKAP and PSD-95 complex (Naisbitt et al., 1999, supra). Shank usesdistinct domains to bind to GKAP and to Homer, and thus can form abridge between proteins of this family. Shank/GKAP is also associatedwith NMDA

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It is to be understood that while the invention has been described inconjunction with the detailed description thereof, the foregoingdescription is intended to illustrate and not limit the scope of theinvention, which is defined by the scope of the appended claims. Otheraspects, advantages, and modifications are within the scope of thefollowing claims.

TABLE 1 Data Collection, Phase Calculation, and Refinement StatisticsMAD Data Collection Statistics Wavelength (Σ) 09879 09793 09790 09611Unique reflections 24051 24179 24226 24481 Redundancy 6.2 6.2 6.2 6.3Completeness (%) 96.8 97.2 97.3 98.4 Signal (<I>/[<I>)^(a) 21.5 21.020.9 20.4 (2.3) (2.1) (2.0) (1.9) R_(sym) (%) 8.1 8.7 9.1 8.8 Overallfigure of merit 0.71 MAD Structure Factor Ratios^(b) and AnomalousScattering Factors^(c) Wavelength (Σ) 0.9879 0.9783 0.9790 0.9611 0.98790.033 0.040 0.032 0.026 0.9793 0.047 0.029 0.044 0.9790 0.063 0.0360.9611 0.050 f′ (e) −4.87 −9.96 −8.06 −4.15 f″ (e) 0.47 3.77 6.28 4.12Refinement Statistics R_(crys)t (%) 25.3 R_(free) (%) 28.4 Average B(X²) 24.8 protein/ 31.7 solvent  No. of water molecules 88 RMSD bondlengths (X) 0.0126 RMSD bond angles (°) 1.745 RMSD B values (X²)0.837/1.487 bonds/angles main chain 1.021/1.594 bonds/angles side chains^(a)Values in parentheses are for the highest resolution shell(1.73-1.70×). R_(sym) = 100 × Σ | I − <I> |/ΣI where I is the integratedintensity of a given reflection. ^(b)RMS (Δ | F |)/RMS (| F |) where Δ |F | is the Bijvoet difference at one wavelength (values on the diagonal)or the dispersive differences between two wavelength (values off thediagonal). ^(c)Anomalous components of the Se scattering factors as afunction of wavelength as determined by SOLVE (Terwilliger andEisenberg, 1983). ^(d)All rounds of refinement included data for which |F | >2.0[. R value = Σ | F_(p)(obs) − Fp(calc) |/ΣF_(p)(obs), whereF_(p) is the structure factor amplitude. The free R value was calculatedfrom 10% of the data that was excluded from the refinement (Brünger,1992). Amino Acid Residues and the Homer Binding Domain Expression LevelMutation (Western Blot) Binding^(a) Homer 2 EVH WT ++ +− F7A − NDF7R + + S8L + − N23A ++ + S28A + − V34M ++ + S35V ++ + D39A ++ − R42E ++− R42A ++ + R46A ++ − R46C ++ + I48A ++ + N58A ++ + N64G ++ + F67S + −K69A ++ + Q72A ++ + F74A ++ + F74L ++ + F90S ++ + E93K + + H95A ++ +L96S + + F109C ++ + ^(a)(−) indicates substantially reduced bindingrelative to wild-type (+).

TABLE 2 WASP EVH1 Mutations WASP Residue/Mutation Homer Residue Table2A- θ1 region Exposed L39M Met 1 C43W Pro 5 L46P Ser 8 T48I Arg 10 E133KHis 95 Buried/partially buried T45M Phe 7 A47D Thr 9 A49E Ala 11 Table2B - θ3 region Exposed S82P/F Arg 42 Buried/partially buried F84L Val 44R86C/H/P/L Arg 46 G89D Ser 49 Table 2C - Other mutations Exposed P58LPro 18 E131K Glu 93 Buried/partially buried H68P Ser 28 V75M Ser 35Y107S/C Phe 67 G125R Gly 87 F128S Phe 90 A134T/V Leu 96 Other A56V —W97C —

Homer Sequence Listing SEQ ID No. Sequence 1 Human Homer 1a (nucleicacid) 2 Human Homer 1a (amino acid) 3 Human Homer 1b (nucleic acid) 4Human Homer 1b (amino acid) 5 IRS-1 6 β-spectrin 7 Human Homer 2a(nucleic acid) 8 Human Homer 2a (amino acid) 9 Human Homer 2b (nucleicacid) 10 Human Homer 2b (amino acid) 11 Human Homer 3 (nucleic acid) 12Human Homer 3 (amino acid) 13 peptide binding-core region: PPXXFR 14peptide binding-extended region: ALTPPSPFRD 15 Homer interactingprotein: rat I30 (nucleic acid) 16 Homer interacting protein: rat I30(amino acid) 17 Homer interacting protein: rat I42 (nucleic acid) 18Homer interacting protein: rat I42 (amino acid) a-b 19 Homer interactingprotein: human I30 (nucleic acid) 20 Homer interacting protein: humanI30 (amino acid) 21 Homer interacting protein: human I42 (nucleic acid)22 Homer interactin protein: human I42 (amino acid) a-c 23 Mouse Homer1a (nucleic acid) 24 Mouse Homer 1a (amino acid) 25 Mouse Homer 1b(nucleic acid) 26 Mouse Homer 1b (amino acid) 27 Mouse Homer 2a (nucleicacid) 28 Mouse Homer 2a (amino acid) 29 Mouse Homer 2b (nucleic acid) 30Mouse Homer 2b (amino acid) 31 Mouse Homer 3 (nucleic acid) 32 MouseHomer 3 (amino acid) 33 Rat Homer 1a (nucleic acid) 34 Rat Homer 1a(amino acid) a-c 35 Rat Homer 1b (nucleic acid) 36 Rat Homer 1b (aminoacid) 37 Rat Homer 1c (nucleic acid) 38 Rat Homer 1c (amino acid) a-b 39Rat Shank 3a (nucleic acid) 40 Rat Shank 3a (amino acid) a-d 41 HumanHomer 3a (nucleic acid) 42 Human Homer 3a (amino acid) 43 Rat INADLpartial nucleic acid sequence 44 Rat INADL partial amino acid sequence47 optimal ligand for Mena: FPPPP 48 ligand motif: KIAA 49 forwardprimer for PCR: 5′-GACAGCAGAGCCAACACCGTG-3′ 50 reverse primer for PCR:5′-GTCTGCAGCTCCATCTCCCAC-3′ 51 primer for PCR:5′-CACGGTGTTGGCTCTGCTGTC-3' 52 degenerate primer:5′-ATGGG(A/G/C)GA(A/G)CA(A/G)CC(T/C/G)AT(T/C)TTC-3′ 53 conserved aminoacid sequence: MGEQPIF 54 oligonucleotide for PCR:5′-GAGGGTAGCCAGTTCAGCCTC-3′ 55 oligonucleotide for PCR:5′-GTTGATCTCACTGCATTGTTC-3′ 56 peptide contained in Homer 1b and 1c:IFELTELRDNLAKLLECS 57 peptide contained in Homer 2a and 2b:GKIDDLHDFRRGLSKLGTDN 58 peptide contained in Homer 3:RLFELSELREGLARLAEAA 59 Homer residues 1307-1316: LVPPPEEFAN 60 Homerresidues 1563-1572: PLPPPLEFSN 61 Homer residues 813-822: PLPPPLEFAN 62Homer residues 878-887: FLPPPESFDA

1. An isolated protein encoded by the nucleic acid sequence as set forthin SEQ ID NO:7.
 2. An isolated protein comprising the amino acidsequence as set forth in SEQ ID NO:1.